Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
Rotavirus Enterotoxin NSP4 and Methods of Using Same
This work was supported in part by Public Health Service Award DK 30114 from
the
National Institutes of Health.
FIELD OF THE INVENTION
This invention relates to the viral enterotoxin NSP4 and to methods for using
it, or
antibodies/antisera thereto, as diagnostic agents, vaccines and therapeutic
agents for the
detection, prevention and/or treatment of rotaviral disease, for the
prevention of stunted
growth in animals and children caused by rotaviral infection and for the
treatment of cystic
fibrosis. This invention also relates to methods and animal models for 1) the
screening for
viral enterotoxins, 2) the detection of viral enterotoxins and 3) the
identification of viral
enterotoxins.
BACKGROUND OF THE INVENTION
Rotaviruses are the leading cause of severe, life-threatening viral
gastroenteritis in
infants and animals (I~apikian et al., 1996) and are associated with sporadic
outbreaks of
diarrhea in elderly (Halvorsrud 1980) and immunocompromised patients (Holzel
et al.,
1980). These viruses have a limited tissue tropism, with infection primarily
being restricted
to cells of the small intestine (Estes et al., 1994). Rotavirus infections
also cause morbidity
and mortality in many animal species. Moreover, the outcome of infection is
age-related;
although rotaviruses may infect individuals and animals of all ages,
symptomatic infection
(i.e., diaxrhea) generally occurs in the young (6 months - 2 years in
children, and up to 14
days in mice), and the elderly.
Age-related host factors which may influence the outcome of infection have
been
proposed to include 1) differences in the presence/quantity of virus-binding
receptors on
mature villas epithelial cells, 2) virus strains with a specific spike protein
(VP4), 3) passive
immunity acquired by maternal antibody or in colostrum, and 4) reduced levels
of proteases
in the young.
Disease resulting from rotavirus infection in mice has been studied more
extensively
than in any other species and an age restriction of disease has been reported
by several
investigators (Ramig 1988; Wolf et al. 1981; Riepenhoff Talty et al., 1982).
Only mice less
than 14 days of age develop diarrhea following oral inoculation of marine
rotavirus, and the
1
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
peak age at which animals are most likely to develop diarrhea (6-11 days)
corresponds to the
age when rotavirus can bind to mouse enterocytes (Riepenhoff Talty et al.,
1982). Treatment
of 8 day old mice with cortisone acetate which promotes premature maturation
of intestinal
epithelial cells, results in a reduced susceptibility to rotavirus-induced
diarrhea, although the
mice can still be infected (Wolf et al., 1981). These data were interpreted to
suggest that the
capacity of marine rotaviruses to induce diarrhea in young, but not adult
mice, is due to the
quantity of rotavirus-binding receptors on the surface of villas epithelial
cells in the young
mouse intestine.
When compared to rotavirus infections in other species, rotavirus infections
in mice
show minimal histologic alterations. That is, villas blunting is limited and
transient, and
crypt cell hyperplasia is not present. In addition, the Ioss of villas tip
epithelial cells is more
limited in mice than in other animals. Instead, vacuolization of enterocytes
on the villas tips
is a predominant feature in symptomatic rotavirus infection in mice and virus
replication may
be abortive (Greenberg et al., 1981). The lack of extensive pathologic
alterations in the
mouse intestine during synptomatic infections has remained a puzzle; one
interpretation of
this phenomenon is that a previously unrecognized mechansm of diarrhea
induction may be
active in symptomatic rotavirus infection in mice.
Despite the prevalence of rotavirus infections and extensive studies in
several animal
models and many advances in understanding rotavirus immunity, epidemiology,
replication
and expression, rotavirus pathogenesis, specifically, the mechanism of
diarrhea induction,
remains poorly understood. Proposed pathophysiologic mechanisms by which
rotaviruses
induce diarrhea following viral replication and viral structural protein
synthesis include
malabsorption secondary to the destruction of enterocytes (Graham et al.,
1984), disruption
of transepithelial ion homeostasis resulting in fluid secretion (Collins et
al., 1988), and local
villas ischemia leading to vascular damage and diarrhea (Osborne et al.,
1988). However,
these proposed mechanisms do not explain cases of rotavirus-induced diarrhea
observed prior
to, or in the absence of, histopathologic changes (Theil et al., 1978;
McAdaragh 1980; Saif et
al., 1976).
On the other hand, the pathophysiology of bacterial-induced diarrhea based on
interactions with intestinal receptors and bacterial enterotoxins is well
understood (Barges et
al., 1978; Gianella et al., 1981; Krause et al., 1990). The heat-stable toxin
A and the heat-
labile toxin of E. coli, and guanylin (an endogenous, 15 amino acid intestinal
ligand
2
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
originally isolated from rat jejunum) induce diarrhea by binding a specific
intestinal receptor,
increasing cAMP or cGMP, and activating a cyclic nucleotide signal
transduction pathway
(Giannella et al., 1993; Currie et al., 1992; Field et al., 1978; Forte et
al., 1992). The net
effect of these bacterial toxins is to increase Cl- secretion, and decrease Na
and water
S absorption.
Previous studies in insect cells indicated that a receptor-mediated
phospholipase C
pathway is associated with the increases in [Calf];, following exogenous
treatment of cells
with NSP4 or NSP4 114-13S peptide (Tian et al., 1994). The rotavirus
nonstructural ER
glycoprotein, NSP4, has been shovcnz to have multiple functions including the
release of
calcium from the endoplasmic reticulum (ER) in SF9 insect cells infected with
recombinant
baculovirus .containing the NSP4 cDNA (Tian et al., .1994; Tian et al., 1995).
In addition,
NSP4 disrupts ER membranes and may play an important role in the removal of
the transient
envelope from budding particles during viral morphogenesis. NSP4 114-135, a 22
as peptide
of NSP4 protein, has been shown to be capable of mimicking properties
associated with
1 S NSP4 including being able to (i) mobilize intracellular calcium levels in
insect cells when
expressed endogenously or added to cells exogenously (Tian et al., 1994; Tian
et al., 1995),
and (ii) destabilize liposomes.
Expression of NSP4 in insect cells increased [Ca2+]; levels from a subset of
the
thapsigargin-sensitive store (ER) (Tian et al., 1995). The [Ca2+]; mobilized
by NSP4 or the
NSP4 114-13S peptide added exogenously to cells was blocked by a phospholipase
C
inhibitor, the U-73122 compound, suggesting that a receptor-mediated pathway
is responsible
for the calcium release from the ER induced by NSP4 (Tian et al., 1995). The
[Caz+];
mobilized by NSP4 expressed intracellularly was not blocked by the U-73122
compound,
suggesting that a second pathway is responsible for the calcium release from
the ER induced
2S by intracellular NSP4 (Tian et al., 1995).
SUMMARY OF THE INVENTION
The present invention discloses herein a method of immunization against
rotavirus
infection or disease comprising administering to a subject a peptide NSP4 112-
17S or NSP4
112-1S0 or a toxoid thereof. Further, the present invention discloses a method
of
immunization against rotavirus infection or disease comprising administering
to a subject a
non-glycosylated NSP4 protein or a toxoid of NSP4. The immunizations may
result in both
homotypic and heterotypic immunity.
3
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
In another specific embodiment, it is also provided a method of passive
immtuuzation
against rotavirus infection comprising administering to an expectant mother a
peptide NSP4
112-175, NSP4 112-150 or a toxoid thereof. Yet further, the present invention
discloses a
method of passive immunization against rotavirus infection comprising
administering to an
expectant mother a non-glycosylated NSP4 protein or a toxoid of NSP4. The
immunizations
may result in both homotypic and heterotypic immunity.
A specific embodiment of the present invention is that the NSP4 peptide (e.g.,
NSP4
I 12-175 or NSP4 I 12-150) or toxoid is produced by a synthetic method. In a
further specific
embodiment, the NSP4 peptide or toxoid is produced by an expression vector.
The
expression vector is selected from the group consisting of mammalian, yeast,
bacterial or
insect.
Another embodiment of the present invention is a fusion protein comprising a
protein
that forms a virus-like particle linked to a NSP4 peptide. One skilled in the
art realizes that
the present invention is not limited to one copy of NSP4 peptide, in fact, the
fusion protein
may contain multiple copies of a NSP4 peptide. The fusion protein further
comprises a linker
sequence. An exemplary linker sequence includes, but is not limited to, three
alanine
residues and an alanine and serine residue. It is also contemplated that three
glycine residues
may be substituted for the three alanine residues. One of skill in the art is
cognizant that the
scope of the invention is not limited to a five residue liucer. It is
contemplated that other
linkers may be used, for example, but not limited to, three alanine residues
or 3 glycine
residues. The NSP4 peptide is NSP4 112-175 or a toxoid thereof or NSP4 112-150
or a
toxoid thereof. The protein that forms ~ a virus-like particle is a viral
protein or peptide
isolated from the viral families Calicivi~idae or Reovif°idae.
Iri specific embodiments, the viral protein isolated from Calicivi~idae is a
Norwalk
virus protein or peptide. In two particular fusion proteins, the Norwalk virus
protein is ORF2
or ORF2 plus ORF3 or a fragment or toxoid of ORF2 or ORF2 plus ORF3.
Specifically,
ORF2 comprises amino acids 21-530 and ORF3 comprises 1-212.
In yet another specific embodiment, the viral protein or peptide isolated from
Reovi~idae is a rotavirus protein or peptide. More particularly, the rotavirus
peptide is
selected from the group of rotavirus proteins consisting of VP2, VP4, VP6 and
VP7. In
4
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
specific embodiments, the rotavirus peptide is VP2 or a VP2 fragment.
Specifically VP2
comprises amino acids 94-881.
Another embodiment of the present invention discloses an expression vector
comprising a nucleic acid sequence encoding a fusion protein operatively
linked to a first
promoter sequence, and a nucleic acid sequence encoding a viral peptide that
is part of a
virus-like particle operatively linked to a second promoter sequence. The
fusion protein
comprises a viral peptide that is part of a virus-like particle linked to a
NSP4 peptide or more
than one NSP4 peptide. The viral peptide linked to the NSP4 peptide is VP2 and
forms the
inner shell of the virus-like particle. The viral peptide that is not linked
to the fusion protein
is rotavirus VP6. This peptide forms the outer shell of the virus-like
particle surrounding the
VP2 shell. The first promoter sequence is a polyhedrin promoter sequence and
the second
promoter sequence is a p10 promoter sequence. The expression vector is
selected from the
group consisting of insect, mammalian, viral and bacterial
In specific embodiments, the expression vector comprises a fusion protein that
comprises a nucleic acid sequence encoding rotavirus VP2 amino acids 94-881
linked to a
nucleic acid sequence encoding NSP4 amino acids 112-175.
In another embodiment, the expression vector comprises a fusion protein that
comprises a nucleic acid sequence encoding rotavirus VP2 amino acids 94-881
linked to a
nucleic acid sequence encoding NSP4 amino acids 112-150.
Another embodiment of the present invention comprises an expression vector
comprising a nucleic acid sequence encoding a fusion protein, wherein the
fusion protein
comprises a viral peptide that is part of a virus-like particle linked to a
NSP4 peptide. The
nucleic acid sequence is operatively linked to a promoter sequence.
In specific embodiments, the expression vector comprises the fusion protein
that
comprises a nucleic acid sequence encoding Norwallc virus ORF2 linked to a
nucleic acid
sequence encoding NSP4 amino acids 112-175. In a further embodiment, the
fusion protein
comprises a nucleic acid sequence encoding Norwalk virus ORF2 linked to a
nucleic acid
sequence encoding NSP4 amino acids 112-150. It is also contemplated that the
fusion
protein may be a fragment of Norwalk virus ORF2 linked to NSP4 amino acids 112-
175 or a
fragment of Norwalk virus ORF2 linked to NSP4 amino acids 112-150.
5
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
In another embodiment, the expression vector comprises the fusion protein that
comprises a nucleic acid sequence encoding a nucleic acid sequence encoding
Norwalk virus
ORF2 and ORF3 linked to NSP4 amino acids 112-175. In another embodiment the
fusion
protein comprises a nucleic acid sequence encoding Norwalk virus ORF2 and ORF3
linked to
a nucleic acid sequence encoding NSP4 amino acids 112-150. It is also
contemplated that the
fusion protein may be a fragment of Norwalk virus ORF2 and ORF3 linked to NSP4
amino
acids 112-175 or a fragment of Norwalk virus ORF2 and ORF3 linked to NSP4
amino acids
112-150.
A specific embodiment of the present invention also comprises a vaccine for
inducing
the formation of protective antibodies against rotavirus infection comprising
administering a
chimeric virus-like particle. The chimeric virus-like particle comprises a
peptide NSP4 112-
175, a first viral protein that is part of a virus-like particle, and a second
viral protein that is
part of a virus-like particle. Specifically, the first viral protein is
rotavirus VP2, which forms
an inner shell and said second viral protein is rotavirus VP6, which forms an
outer shell
surromding the VP2 shell.
In another embodiment, it is also provided a method of immunization against
rotavirus infection or disease comprising the step of administering to a
subject a compound
comprising a chimeric virus-like particle. The chimeric virus-like particle
comprises a
peptide NSP4 112-175, a first viral protein that is part of a virus-like
particle, and a second
viral protein that is part of a virus-like particle. More particularly, the
first viral protein is
rotavirus VP2 and said second viral protein is rotavirus VP6. The compound is
administered
orally, parenterally or intranasally. Another aspect comprises that the
compound is
administered with an adjuvant.
In specific embodiments, the compound is simultaneously or consecutively
administered by at least two different routes of administration. Exemplary
routes of
administration include, but are not limited to, oral, parenteral or
intranasal.
Another embodiment of the present invention comprises a method of inducing an
immune response comprising the step of administering to a mammal one
expression vector,
wherein said expression vector comprises a nucleic acid sequence encoding a
fusion protein,
wherein said fusion protein comprises a first viral protein that is part of a
virus-like particle
linked to a NSP4 nucleic acid sequence, and a nucleic acid sequence encoding a
second viral
6
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
protein that is part of a virus-like particle. In specific embodiments, the
nucleic acid
sequence encoding the fusion protein and the nucleic acid sequence encoding
the second viral
protein are under separate transcriptional control and wherein the nucleic
acid sequence
encoding the fusion protein and the nucleic acid sequence encoding the second
viral protein
are in tandem in the one expression vector.
A specific embodiment also provides a method of inducing an immune response
comprising the steps of co-administering to a mammal or a cell two different
expression
vectors, wherein a first expression vector comprises a nucleic acid sequence
encoding a first
viral protein that is part of a virus-like particle and a second expression
vector comprises a
nucleic acid sequence encoding a fusion protein, wherein said fusion protein
comprises a
second viral protein that is part of a virus-like particle linked to a NSP4
nucleic acid
sequence.
Other embodiments, features and advantages of the present invention will
become
apparent from the following detailed description. It should be understood,
however, that the
detailed description and the specific examples, while indicating preferred
embodiments of the
invention, are given by way of illustration only, since various changes and
modifications
within the spirit and scope of the invention will become apparent to those
skilled in the art
from this detailed description.
DESCRIPTION OF THE DRAWINGS
The following drawings form part of the present specification and are included
to
further demonstrate certain aspects of the present invention. The invention
may be better
understood by reference to one or more of these drawings in combination with
the detailed
description of specific embodiments presented herein.
FIG. 1 shows rotavirus NSP4 protein induces diarrhea in CDl mice. 0.1 to 5
nmols
(2-100 p,M) of purified NSP4 was administered by the IP or IL routes to 6-7, 8-
9 and 17-18
day old CDl pups. Rotavirus protein VP6 was used as the control in 6-7 day old
animals, the
most sensitive. The dose and route of the proteins, age of the animals, and
mean diarrhea
score (mean score) are indicated on the bottom of the graph. The Y axis
displays the
diarrhea. Above each column is the number of responders (mice with diarrheal
disease) over
the total number of animals tested.
7
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
FIG. 2 shows IP administration of NSP4 induces diarrhea in 6-7 day old CD-1
pups.
0.04 to 1.0 nmols (1-25 fig) of purified NSP4 protein was administered to 6-7
day old CDl
pups by the IP route. The dose administered, in nanomoles and in micrograms,
is shown on
the X-axis. The percentage of pups that display diarrhea in response to the
administered dose
is shown on the Y-axis. Above each column the number of responders (pups with
diarrhea)
over the total number of animals receiving treatment is shown.
FIG. 3 shows diarrhea) response in CD1 mice following IP administration of
NSP4
114-I35 peptide. Young (6-7 day) mouse pups were injected with various doses
of peptide (x
axis) and monitored for disease. The number of responders over the total
number animals
tested is shown above each column. With the CDI mice, 0.1 - 50 nmol (2~,M-1mM)
of
peptide elicited similar responses (30-40% diarrhea induction); and 100-400
nmols (2-8~
of peptide elicited comparative responses with 60-70% of the animals siclc.
FIG. 4 shows diarrhea) response in Balb/C mice following IP administration of
NSP4
114-135 peptide. Young (6-7 day) mouse pups were injected with various doses
of peptide (x
axis) and monitored for disease. The number of responders over the total
number animals
tested is shown above each column.
FIG. 5 shows IP delivery of NSP4 114-135 peptide induce an age-dependent
diarrhea
in CD1 mice and Sprague-Dawley Rats. Different age outbred mice and rats were
inoculated
(IP) with NSP4 114-135 peptide and evaluated for disease. The age and species
of the pups,
dose of the synthetic peptide and indication of whether peptides were unlinked
or cross-
linked are indicated on the bottom of the graph. The dose of the IP delivered
peptide varied
with the age of the animals, r. e., older animals received a higher dose to
control for the
differences in body weight. The Y axis indicates the % diarrhea and above each
column is
indicated the number of responders over the total number of animals
inoculated. The peptide
was diluted in sterile PBS and evaluated for sterility. A final volume of 50
~l per dose was
used.
FIG. 6 shows IL delivery of NSP4 114-135 peptide induce an age-dependent
diarrhea
in CD1 mice and Sprague-Dawley Rats. Different age outbred mice and rats were
inoculated
(IL) with NSP4 114-135 peptide and evaluated for disease. The age and species
of the pups,
dose of the synthetic peptide and indication of whether peptides were unlinked
or cross-
linked are indicated on the bottom of the graph. The Y axis indicates the %
diarrhea and
8
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
above each column is indicated the number of responders over the total number
of animals
inoculated. The peptide was diluted in sterile PBS and evaluated for
sterility. A final volume
of 50 ~.1 per dose was used.
FIG. 7A and FIG. 7B shows the dose response for cross-linked NSP4 peptide
delivered ip to 6-7 day old CD1 and Balb/C Mice. The NSP4 114-135 peptide was
cross-
Iinl~ed to itself with glutaraldehyde a~ld dialyzed against sterile PBS prior
to IP delivery to
CD1 (FIG. 7A) and Balb/C (FIG. 7B) mice. The number of responders over the
total number
of animals inoculated is shown above each column.
FIG. 8 illustrates the experimental designs to test the ability of NSP4 114-
135 to
induce protective immunity from infectious rotavirus challenge and the ability
of NSP4-
specific antibody to mitigate rotavirus diarrhea following infection. The left
hand side of the
figure illustrates that mouse dams were immunized with NSP4 peptide or With
control
peptide and then bred. Pups born to the dams were orally challenged with
virulent rotavirus
at 6-7 days. The right hand side of the figure illustrates how pups born to
non-immunized
dams were first orally challenged with virulent rotavirus, followed by oral
gavage of NSP4-
specific or control antisera. The results of these experiments are set forth
in Tables 5 and 6.
FIG. 9 shows the results from an experiment to study the differences in weight
and
growth between normal animals and animals suffering from NSP4 I 14-135-induced
diarrhea.
FIG. 10 shows the amino acid sequence comparison of NSP4 from OSU attenuated
and OSU virulent virus. The amino acid sequence of the NSP4 protein of OSU-a
(a porcine
rotavirus, tissue culture attenuated, avirulent strain, SEQ.ID.N0:7)" top
line, is compared to
the amino acid sequence of the NSP4 protein of OSU-v (a porcine rotavirus,
virulent strain,
SEQ.ID.N0:8), bottom line. Positions at which the two sequences are different
are shown in
bold.
FIG. 11A and FIG. 11B show purity of NSP4 112-175 and its interaction with an
antisenun raised with synthetic SAl l NSP4 peptide 114-135. FIG. 11A
illustrates the silver
stained SDS-15% polyacrylamide gel of purified NSP4 112-175. FIG. 11B
illustrates the
Western blot of purified NSP4 112-175 tested by rabbit antiserum against
synthetic SA11
NSP4 peptide 114-135 (1:300 dilution). Lane l, NSP4 112-175 crude material.
Lane 2,
eluate from immune affinity column against baculoviral proteins. Lane 3, 0.5
~,g of purified
NSP4 112-175. Arrows indicate NSP4 112-175.
9
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
FIG. 12A and FIG. 12B show the senun antibody responses in dams and pups to
parenterally administered NSP4 112-175. Mice were immunized three times with
15 ~,g of
NSP4 aa112-175 plus 20 ~,g of QS-21 or with 20 ~,g of QS-21 alone. Blood
samples were
collected at 0, 7, and 16 DPP. FIG. 12A shows the serum antibody titers
against NSP4 1 I2-
175. FIG. 12B shows the Serum antibody titers against full-length NSP4. Bars:
NSP4 dams,
immunized with NSP4 112-175 plus QS-21; NSP4-pups: delivered to an nursed by
NSP4-
dams; QS-21-dams, inoculated with QS-21 alone; QS-21-pups: delivered to and
nursed by
QS-21-dams.
FIG. 13A and FIG. 13B show the serum antibody responses in pups during cross-
nursing. FIG. 13A illustrates the serum antibody titers to NSP4 112-175 in
pups. FIG. 13B
illustrates the serum antibody titers to full-length NSP4 in pups. Blood
samples were
r
collected on 0, 7, and 16 DPP.
FIG. 14A, FIG. 14B and FIG. 14C show replication of simian rotavirus SAll in
seven-day-old mice. SA11, at a dose of 20 DDSO was orally gavaged in seven-day-
old
BALB/c mice born to NSP4 dams with QS-21 dams. Titers of infectious virus in
the
combined small and large intestine homogenates were determined at various days
postinfection by plaque assay. The limit of virus detection was about 50
PFU/ml. -~-,
virus titers in intestinal homogenates from QS-21-pups. -o-virus titers in
intestinal
homogenates from NSP4-pups. -~-, baseline of intestines from pups with mock
infection. FIG 14A and FIG. 14B show the virus titers from 2 individual
experiments and
each experiment was composed of one inoculated and one control pup 1-7 DPI.
FIG. 14C
shows the average virus titers given in FIG. 14 A and FIG. 14B.
FIG. 15 shows serum antibody responses against SA11 VP6 in dams and pups with
or
lacking antibody to NSP4 112-175 and challenged with a single dose of
rotavirus SA11.
Pups were challenged with a single dose of 20 DDso of SAl 1. Blood samples
were collected
on 0 DPI and 12 DPI. Bars: Bars: NSP4 dams, immunized with NSP4 112-175 plus
QS-21;
NSP4-pups: delivered to an nursed by NSP4-dams; QS-21-dams, inoculated with QS-
21
alone; QS-21-pups: delivered to and nursed by QS-21-dams. n, number of mice
per group.
The antibody GMT between NSP4-pups and QS-21-pups were 3000 vs. 300.
FIG. 16 shows a Western blot analysis of NSP4-2/6-VLPs. A fusion protein was
constructed comprising NSP4 112-175 and as 94-881 of VP2. The fusion was
inserted into a
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
baculovirus expression vector pBAC4x-I. Cells were transiently transfected
with the
expression vector. Virus-like particles were expressed, purified, and analyzed
by Western
blot.
FIG. 17 shows the effect of NSP4 on transepithelial resistance (TER) of MDCK-1
cells and neutralization by antibody. This is. an in vitro assay that mimics
the effect of
antibody on induction of diarrhea in mice.
DETAILED DESCRIPTION OF THE INVENTION
This invention stems from the discovery of the first known viral enterotoxin,
rotavirus
NSP4, previously called NS28, which encodes a viral toxin capable of inducing
intestinal
secretion through a heretofore unknown signal transduction pathway to cause
diarrheal
disease.
The present invention relates to the fortuitous discovery that the rotavirus
nonstructural ER glycoprotein, NSP4, induces an age-dependent diarrhea in two
rodent
models. Induction of diarrhea following administration of this protein alone
was completely
unexpected because infection with rotavirus was not involved. Characterization
of the
parameters of these new models of rotavirus-induced diarrhea demonstrates that
this enteric
viral-encoded protein is an enterotoxin, similax to bacterial enterotoxins,
which are well-
known to induce diarrhea by stimulating signal transduction pathways following
interaction
with specific intestinal receptors. The ordinary practitioner will appreciate
that these new
findings on NSP4-induced diarrheal disease and the data presented herein
support several
novel therapeutic and preventive approaches to rotavirus-induced disease.
The present invention also demonstrates that a synthetic peptide corresponding
to
amino acids 114.-135 of SA11 NSP4 also induces an age-dependent diarrhea in
young mice
comparable to NSP4 when administered by the IP and IL route. Since the NSP4
114-135
peptide was readily available in large amounts in pure form, the response to
the peptide was
studied in detail. The response to the peptide was specific as shown by 1)
lack of response to
control peptides, 2) blocking with peptide-specific antibody, and 3) a mutated
peptide
(differing by only a single residue) alone failed to induce the response. The
concentration of
peptide required for disease induction was considerably higher than that
needed for a
response to the protein. Because the entire protein possesses more potent
activity than the
peptide, other peptides from this protein which have the same effect are also
included in the
11
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
present invention. These peptides include, but are not limited to, NSP4, non-
glycosylated
NSP4, NSP4 114-135, 120-147, 112-175 or 112-150. Also contemplated in the
present
invention is the use of toxoids of the above listed NSP4 proteins or peptides.
Specifically, proteins and or peptides according to the present invention may
contain
the entire amino acid sequence of NSP4 protein or any other fragment of NSP4
as set forth
herein. The following amino acid sequences are sequences corresponding to NSP4
proteins
and are within the scope of the invention and some are referenced with the
corresponding
GenBank Accession Numbers
(http://www.ncbi.nlm.nih.gov/Genbank/GenbankSearch.htinl):
SAll (SEQ.ID.N0:11, AAC61867); SA11 clone 3 (SEQ.ID.N0:12); Murine EC
(SEQ.ID.N0:13, AAB58700); Porcine OSU (SEQ.ID.N0:7, BAA13728); Gott-v
(SEQ.ID.N0:14) and Gott-a (SEQ.ID.NO:15).
Further, the present invention illustrates an analogous age dependence in the
induction
of diarrhea with purified NSP4 protein and NSP4 114-135 peptide. Mice were
most sensitive
to the effects of the protein or peptide at 6-7 days of age. Diarrhea
induction by NSP4 or
NSP4 114-135 decreased as the age of the animal increased, regardless of the
route of
administration. Hence the observed diarrhea in this study mimics the
properties of
symptomatic infection observed in experimental and natural rotavirus
infection.
The present invention also shows that the inoculation of NSP4 114-135 peptide-
specific antiserum prior to IP delivery of peptide results in a dramatic
reduction of disease.
(90% reduction in disease).
Further, the present invention shows that diarrheal disease in pups born to
dams
immunized with the NSP4 114-135 and NSP4 112-175 peptide is significantly
reduced in
severity, duration, and in the number of pups with diarrhea. Further, the
present invention
demonstrates that immunization of dams with highly pure NSP4 112-175 induces
passive
protection of suckling mice against diarrhea induced by challenge viruses
homotypic or
heterotypic to the NSP4 112-175 immunogen.
Yet further, post-infection administration of NSP4-specific antibody
significantly
reduces diarrheal disease.
One skilled in the art realizes that these data, showing that NSP4 protein or
NSP4
114-135 and NSP4 112-175-specific antibodies' are sufficient to block the
induction or
12
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
severity of diarrhea, demonstrate that NSP4, NSP4 114-135 or NSP4 112-175
and/or
antibodies specific thereto will be useful as vaccines and as therapeutic
agents. Additionally,
new drugs can now be developed to block or minimize the effects of interaction
of NSP4 with
its receptor or the effects of the disruption of the calcium homeostasis in
affected cells.
In accordance with the foregoing and with the disclosure that follows, it is
an
embodiment of the present invention to provide a method for the screening and
identification
of viral enterotoxins associated with rotavirus and other gastroenteritis
viruses, such as
caliciviruses, astroviruses, enteric adenoviruses, coronaviruses, and
parvoviruses, including
administering expressed proteins or peptides or synthetic peptides of such
viruses to animals
and monitoring the animals for diarrhea. For the purpose of this and other
embodiments of
the invention, human volunteers shall be considered to be within the scope of
"animals." It is
a further embodiment of the present invention to provide methods for the
screening and
identification of new viral enterotoxins including administration of expressed
proteins or
peptides or synthetic peptides to CDl mice, Balb/C mice andlor Sprague-Dawley
rats and
monitoring for diarrhea.
To the extent that the meaning of the term "diarrhea-genie viral protein" may
be
construed to differ from the meaning of the term viral enterotoxin, if the
term "diarrhea-genie
viral protein" is substituted for the term viral enterotoxin, the subject
matter of this and all
following embodiments and all claims is also considered to be within the scope
of the
invention, and fully described for all purposes.
It is another embodiment of the present invention to provide a method for the
screening for and identification of viral enterotoxins associated with viruses
that are not
known as diarrhea or gastroenteritis virus, but that are associated with
diarrhea as a
consequence of infection. Without limiting the invention, examples include
human
ixmnunodeficiency virus (HIV) and cytomegalovirus (CMV). This method includes
administering expressed proteins or peptides or synthetic peptides of a
selected virus to
axlimals and monitoring the animals for diarrhea.
In certain embodiments, the present invention provides antibodies that bind
with high
specificity to NSP4 or fragments thereof (i.e., NSP4 114-135, NSP4 120-147,
NSP4 112-175
or NSP4 112-150) provided herein. As detailed above, in addition to antibodies
generated
13
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
against NSP4, antibodies may also be generated in response to smaller
constructs comprising
epitopic core regions, including wild-type and mutant epitopes.
It is another embodiment of the present invention to provide a method for
treatment of
diarrheal disease, including reducing the severity of diarrhea caused by viral
infection,
including administering antibodies to viral enterotoxins to a subject with
diarrhea or known,
or suspected to be infected by or to have been exposed to a gastroenteritis
virus. For the
purpose of this invention, antibodies shall mean polyclonal and monoclonal
antibodies unless
otherwise indicated. Methods for the preparation of polyclonal and monoclonal
antibodies to
any protein or peptide are well known to the practitioner having ordinary
skill in the art.
The term "antibody" is used to refer to any antibody-like molecule that has an
antigen
binding region, and includes antibody fragments such as Fab', Fab, F(ab')2,
single domain
antibodies (DABS), Fv, scFv (single chain Fv), and the like. The techniques
for preparing
and using various antibody-based constructs and fragments are well known in
the art. Means
for preparing and characterizing antibodies are also well known in the art
(See, e.g..,
Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988;
incorporated herein
by reference). Generally, IgG and/or IgM are preferred because they are the
most common
antibodies in the physiological situation and because they are most easily
made in a
laboratory setting. Another antibody that is important for mucosal protection
is IgA. IgA
functions as the primary antibody that is present in body secretions, such as
saliva, tears,
breast milk, gastrointestinal secretions and mucus secretions of the
respiratory and
genitourinary tracts.
"Humanized" antibodies are also contemplated, as are chimeric antibodies from
mouse, rat, or other species, bearing human constant and/or variable region
domains,
bispecific antibodies, recombinant and engineered antibodies and fragments
thereof.
Antibodies, both polyclonal and monoclonal, specific for isoforms of antigen
may be
prepared using conventional immunization techniques, as will be generally
known to those of
skill in the art. A composition containing antigenic epitopes of the NSP4
protein, peptide or
fragments thereof of the present invention can be used to immunize one or more
experimental
animals, such as a rabbit or mouse, which will then proceed to produce
specific antibodies
against the compounds of the present invention. One slcilled in the art
realizes that fragments
of NSP4 protein include, but are not limited to NSP4 114-135, NSP4 120-174,
NSP4 112-175
14
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
or NSP4 112-150. Polyclonal antisera may be obtained, after allowing time for
antibody
generation, simply by bleeding the animal and preparing serum samples from the
whole
blood.
Briefly, a polyclonal antibody is prepared by immunizing an animal with an
immunogen comprising a NSP4 polypeptide or fragment thereof (i.e., NSP4, NSP4
114-135,
NSP4 120-174, NSP4 112-175 or NSP4 112-150) and collecting antisera from that
immunized animal. A wide range of animal species can be used for the
production of
antisera. Typically an animal used for production of anti-antisera is a non-
human animal
including rabbits, chickens, mice, rats, hamsters, pigs or horses. Because of
the relatively
large blood volume of rabbits, a rabbit is a preferred choice for production
of polyclonal
antibodies.
It is another embodiment of the present invention to provide monoclonal
antibodies to
NSP4 protein, to NSP4 114-135 peptide, to NSP4 120-147 peptide, to NSP4 112-
175 peptide,
to NSP4 112-175 and to other peptides of NSP4.
Monoclonal antibodies (MAbs) are recognized to have certain advantages, e.g.,
reproducibility and large-scale production, and their use is generally
preferred. Monoclonal
antibodies may be readily prepared through use of well-knovnm techniques, such
as those
exemplified in U.S. Patent 4,196,265, incorporated herein by reference.
Typically, this
technique involves immunizing a suitable animal with a selected immunogen
composition,
e.g., a purified or partially purified NSP4 protein, polypeptide or peptide or
cell expressing
high levels of NSP4. One skilled in the art realizes that NSP4 protein or.
polypeptide
includes, but is not limited to, NSP4 114-135, NSP4 120-174, NSP4 112-175 or
NSP4 112-
150. The immunizing composition is administered in a manner effective to
stimulate
antibody producing cells. Rodents such as mice and rats are preferred animals,
however, the
use of rabbit, sheep, human, monkey, rabbit, chicken, chiclcen eggs or frog
cells is also
possible. The use of rats may provide certain advantages (Goding 1986), but
mice are
preferred, with the BALB/c mouse being most preferred as this is most
routinely used and
generally gives a higher percentage of stable fusions. '
It is proposed that the monoclonal antibodies of the present invention will
fmd useful
application in standard immunochemical procedures, such as ELISA and Western
blot
methods and in immunohistochemical procedures such as tissue staining, as well
as in other
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
procedures which may utilize antibodies specific to NSP4, NSP4 114-135, NSP4
120-174,
NSP4 112-175 or NSP4 112-150 antigen epitopes. Additionally, it is proposed
that
monoclonal antibodies specific to the particular NSP4 protein or polypeptide
(i.e., NSP4 114-
135, NSP4 120-174, NSP4 112-175 or NSP4 112-150) of different species may be
utilized in
other useful applications.
The present invention also contemplates the use of antibodies against NSP4
proteins,
polypeptides or fragments thereof (i.e., NSP4 114-135, NSP4 120-174, NSP4 112-
175 or
NSP4 112-150), generally of the monoclonal type, that are linked to one or
more other agents
to form an antibody conjugate. Any antibody of sufficient selectivity,
specificity and affinity
may be employed as the basis for an antibody conjugate. Such properties may be
evaluated
using conventional immunological screening methodology known to those of skill
in the art.
In order to increase the efficacy of antibody molecules as diagnostic or
therapeutic
agents, it is conventional to liW~ or covalently bind or complex at least one
desired molecule
or moiety. Such a molecule or moiety may be, but is not limited to, at least
one effector or
reporter molecule. Effector molecules comprise molecules having a desired
activity, e.g.,
cytotoxic activity. Non-limiting examples of effector molecules which have
been attached to
antibodies include toxins, anti-tumor agents, therapeutic enzymes, radio-
labeled nucleotides,
antiviral agents, chelating agents, cytokines, growth factors, and oligo- or
poly-nucleotides.
By contrast, a reporter molecule is defined as any moiety which may be
detected using an
assay. Non-limiting examples of reporter molecules which have been conjugated
to
antibodies include enzymes, radiolabels, haptens, fluorescent labels,
phosphorescent
molecules, cherniluminescent molecules, chromophores, luminescent molecules,
photoaffinity molecules, colored particles or ligands, such as biotin.
Certain examples of antibody conjugates are those conjugates in which the
antibody is
linl~ed to a detectable label. "Detectable labels" are compounds or elements
that can be
detected due to their specific functional properties, or chemical
characteristics, the use of
which allows the antibody to which they are attached to be detected, and
further quantified if
desired. Another such example is the formation of a conjugate comprising an
antibody linked
to a cytotoxic or anti-cellular agent, as may be termed "immunotoxins".
Antibody conjugates are thus preferred for use as diagnostic agents. Antibody
diagnostics generally fall within two classes, those for use in is2 vitro
diagnostics, such as in a
16
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
variety of immunoassays, and those for use ifz vivo diagnostic protocols,
generally known as
"antibody-directed imaging".
Antibody conjugates of the present invention are those intended primarily for
use ih
vbtf"O, where the antibody is linked to a secondary binding ligand or to an
enzyme (an enzyme
tag) that will generate a colored product upon contact with a chromogeuc
substrate.
Examples of suitable enzymes include urease, alkaline phosphatase,
(horseradish) hydrogen
peroxidase and glucose oxidase. Preferred secondary binding.ligands are biotin
and avidin or
streptavidin compounds. The use of such labels is well known to those of skill
in the art in
light and is described, for example, in U.S. Patents 3,817,837; 3,850,752;
3,939,350;
3,996,345; 4,277,437; 4,275,149 and 4,366,241; each incorporated herein by
reference.
Among the fluorescent labels contemplated for use as conjugates include Alexa
350,
Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G,
BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5,6-FAM, Fluorescein
Isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green
514,
Pacific Blue, REG, Rhodamine Green, Rhodamine Red, Renographin, ROX, TAMRA,
TET,
Tetramethylrhodamine, and/or Texas Red.
Yet another known method of site-specific attachment of molecules to
antibodies
comprises the reaction of antibodies with hapten-based affinity labels.
Essentially, hapten-
based affinity labels react with amino acids in the antigen binding site,
thereby destroying
this site and blocl~ing specific antigen reaction.
Molecules containing azido groups may also be used to form covalent bonds to
proteins through reactive nitrene intermediates that are generated by low
intensity ultraviolet
light (Potter & Haley, 1983). In particular, 2- and 8-azido analogues of
purine nucleotides
have been used as site-directed photoprobes to identify nucleotide binding
proteins in crude
cell extracts (Owens & Haley, 1987; Atherton et al., 1985). The 2- and 8-azido
nucleotides
have also been used to map nucleotide binding domains of purified proteins
(Khatoon et al.,
1989; King et al., 1989; and Dholakia et al., 1989) and may be used as
antibody binding
agents.
Several methods are known in the art for the attachment or conjugation of an
antibody
to its conjugate moiety. Some attachment methods involve the use of a metal
chelate
complex employing, for example, an organic chelating agent such a
17
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
diethylenetriaminepentaacetic acid anhydride (DTPA);
ethylenetriaminetetraacetic acid; N-
chloro-p-toluenesulfonamide; and/or tetrachloro-3a-6a-diphenylglycouril-3
attached to the
antibody (IJ.S. Patent Nos. 4,472,509 and 4,938,948, each incorporated herein
by reference).
Monoclonal antibodies may also be reacted with an enzyne in the presence of a
coupling
agent such as glutaraldehyde or periodate. Conjugates with fluorescein markers
are prepared
in the presence of these coupling agents or by reaction with an
isothiocyanate. In U.S. Patent
No. 4,938,948, imaging of breast tumors is achieved using monoclonal
antibodies and the
detectable imaging moieties are bound to the antibody using linkers such as
methyl-p-
hydroxybenzimidate or N-succinimidyl-3-(4-hydroxyphenyl)propionate.
In other embodiments, derivatization of immunoglobulins by selectively
introducing
sulfhydryl groups in the Fc region of an immunoglobulin, using reaction
conditions that do
not alter the antibody combining site are contemplated. Antibody conjugates
produced
according to this methodology are disclosed to exhibit improved longevity,
specificity and
sensitivity (IJ.S. Pat. No. 5,196,066, incorporated herein by reference). Site-
specific
attachment of effector or reporter molecules, wherein the reporter or effector
molecule is
conjugated to a carbohydrate residue in the Fc region have also been disclosed
in the
literature (O'Shannessy et al., 1987). This approach has been reported to
produce
diagnostically and therapeutically promising antibodies which are currently in
clinical
evaluation.
Another embodiment of the present invention is to provide a method of using
NSP4
and the peptides thereof, including but not limited to NSP4 114-135, NSP4 120-
147, NSP4
112-175 and NSP4 112-150 to measure the levels of antibodies to NSP4. These
antibody
measurements rnay be a surrogate for measuring protective immunity. For
example, NSP4
and peptides thereof may be used to measure binding of antibodies, such as in
an ELISA, or
to measure neutralizing antibodies, such as in a transepithelial resistance
assay (TER). Thus,
it is contemplated that these type of in vitoo methods (antibody binding and
antibody
neutralization) may correlate to protective immunity.
It is another embodiment of the present invention to provide a method for the
prevention or amelioration of diarrhea caused by rotavirus infection including
administration
of antibodies to NSP4 protein or peptides thereof, including but not limited
to NSP4 114-135,
NSP4 120-147, NSP4 112-175 and NSP4 112-150. As rotavirus infection is
transmitted
rapidly, this method is considered to include the prevention or amelioration
of disease
18
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
following exposure to a known infected person, for example in day care centers
and in
hospitals.
For the purpose of this invention, the term "compound comprising amino acids
in a
sequence corresponding to NSP4 114-135" shall mean a compound which has within
it a
sequence of amino acids corresponding to the sequence of NSP4 114-135,
including NSP4
1I4-135 and the NSP4 protein. For the pcupose of this invention, the term
"compound
comprising amino acids in a sequence corresponding to NSP4 120-147" shall mean
a
compound which has within it a sequence of amino acids corresponding to the
sequence of
NSP4 120-147, including NSP4 120-147 and the NSP4 protein. For the purpose of
tlus
invention, the term "comprising amino acids in a sequence corresponding to
NSP4 112-175"
shall mean a compound which has within it a sequence of amino acids
corresponding to the
sequence of NSP4 112-175, including NSP4 112-175 and the NSP4 protein. For the
purpose
of this invention, the term "compound comprising amino acids in a sequence
corresponding
to NSP4 112-150" shall mean a compound which has Within it a sequence
corresponding to
the sequence of NSP4 112-150, including NSP4 112-150 and the NSP4 protein. For
the
purpose of this invention, the term "derivative" shall mean any molecules
which are within
the skill of the ordinary practitioner to make and use, which are made by
derivatizing the
subject compound, and which do not destroy the activity of the derivatized
compound.
Compounds which meet the foregoing criteria which diminish, but do not
destroy, the activity
of the derivatized compound are considered to be within the scope of the term
"derivative."
Thus, according to the invention, a derivative of a compound comprising amino
acids in a
sequence corresponding to the sequence of NSP4 114-135, NSP4 120-147, NSP4 112-
175 or
NSP4 112-150 need not comprise a sequence of amino acids that corresponds
exactly to the
sequence of NSP4 114-135, NSP4 120-147, NSP 112-175 or NSP4 112-175 so long as
it
retains a measurable amount of the activity of the NSP4 114-135, NSP4 120-147,
NSP 112-
175 or NSP4 112-150 peptide.
Another aspect of the present invention is a method of irmnunization against
rotavirus
infection or disease comprising admiustering to a subject a peptide NSP4 112-
175 or NSP4
112-150 or a toxoid thereof. The immunization may result in homotypic or
heterotypic
immunity.
It is another embodiment of the present invention to provide a method of
passive
immunization against rotavirus infection comprising administering to an
expectant mother
19
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
NSP4 peptides or toxoids thereof, including, but not limited to NSP4 114-135,
NSP4 120=
174, NSP4 112-17S or NSP4 112-1 S0.
It is also contemplated in the present invention that a non-glycosylated
peptide of
NSP4 may be administered to a subject or to an expectant mother. One skilled
in the art is
S cognizant that NSP4 is a glycosylated protein. The amino terminus of NSP4
contains two N
linked high mannose glycosylation sites, which are located in the first of
three hydrophobic
domains. Glycosylation of NSP4 is required for removal of the transient
envelope from the
budding particles. It is also contemplated that the glycosylation sites may be
mutated using
standard mutagenesis techniques well known and used in the art e.g., site-
directed
mutagenesis.
Inhibition of N-linked glycosylation will be preformed utilizing standard
procedures
that are well known in the art. Exemplary inhibitors of N-linked glycosylation
that may used
include, but are not limited to tunicamycin, deoxynojirimycin,
castanospermine,
deoxymannojirimycin or swainsonine.
1S It is another embodiment of the present invention to provide a method for
the
prevention of decreased growth rates caused by rotavirus infection including
use of the NSP4
protein or peptides thereof, including but not limited to NSP4 114-135, NSP4
120-147, NSP4
112-17S and/or NSP4 112-1S0 as a treatment for or vaccine against rotavirus
diarrhea. In
addition, animals given peptide twice (at a two day interval) showed a rapid
onset of severe
diarrhea followed by stunted growth. The weight of these animals was 20-30%
lower for
three weeks after achninistration of peptide.
The present invention also illustrates that NSP4 114-13S promotes and augments
cAMP-dependent Cl- secretion in mouse intestinal mucosa and induces diarrhea
in rodents in
a time frame similar to STB (about 3 hrs). The electrophysiological data show
that NSP4
2S induces calcium increases in the intestines of mice in an age-dependent
manner and these
increases in calcium result in chloride secretion as measured by short-circuit
currents. Direct
addition of cross-linked NSP4 114-13S to mouse ileal mucosal sheets resulted
in a rise in
current, similar to that evoked by the calcium agonist, carbachol. In addition
to the age-
dependence, induction of chloride secretion from intestinal mucosal sheets was
site-
dependent. Zero to minimal responses were observed when mouse jejunum,
duodenum or
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
colon tissue was employed, and maximum responses were induced when the ileum
was
utilized. These results support the model of NSP4-induced diarrhea.
The present invention also includes, NSP4 112-175, a cleavage product of the
SAl l
NSP4 C-terminus, which was detected in the extracellular medium of rotavirus-
infected cells.
The cleavage product mobilizes calcium and has enterotoxin activity in mice,
similar to full-
length NSP4 (Zhang et al., 2000). Thus, one skilled in the art realizes that
these results
demonstrate that NSP4 is released from virus-infected cells and such
extracellular NSP4
initiates a signaling pathway leading to diarrhea (Ball et al., 1996). These
distinctive
pathogenic effects of NSP4 or NSP4 112-175 suggest that antagonists to NSP4
if2 vivo may
result in protection against rotavirus-induced diarrhea.
These data show that NSP4 stimulation of a Ca2+ -dependent signal transduction
pathway, resulting in disruption of normal intestinal epithelial transport, is
similar to that
reported for guanylin and the heat-stable enterotoxins. Based on the
enteropathogenic
similarities in intestinal secretion with those reported for guanylin and the
heat-stable
enterotoxins, NSP4 can be considered a viral enterotoxin.
It has been demonstrated that NSP4 induces diarrhea by activating an age-
dependent,
calcium-sensitive anion (chloride) permeability in the small and large
intestine mucosa in
both normal mice and mice with cystic fibrosis that lacks the cystic fibrosis
transmembrane
regulation. These properties of NSP4 indicate that it is a novel secretary
agonist since other
secretary agonist fail to function in mice with cystic fibrosis. Further, this
is confirmed in the
present invention. Administration of NSP4, virus or peptide to 5-7 day old
CFTR knock-out
mice (mice homozygous for the mutation in the CFTR chloride channel coding
region that
causes Cystic Fibrosis) results in diarrhea in 100% of the cases.
One of shill in the art is cognizant that the models of NSP4-induced diarrhea
may be
altered. Thus, the scope of the present invention is not limited to one
specific model of
NSP4-induced diarrhea, but includes and is not limited to alterations and
variations of the
model. For example, the data from the Cystic Fibrosis mice suggest that age-
dependence
may be downstream of Ca2+ mobilization, thus a channel may be involved instead
of a
receptor for the regulation of age-dependent disease.
It is another embodiment of the present invention to provide methods for the
screening for and identification of viral enterotoxins associated with
rotavirus and other
21
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
gastroenteritis viruses, such as caliciviruses, astroviruses, enteric
adenoviruses, coronoviruses
and parvoviruses, including in vitro administration of virus, viral proteins
or peptides thereof
to intestinal mucosa tissues or to cells and moutoring chloride secretion
and/or intracellular
calcium levels and/or cAMP levels.
It is another embodiment of the present invention to provide methods for the
screening for and identification of viral enterotoxins associated with other
viruses associated
with diarrhea, including HIV and CMV, including i~z vitro administration of
virus, viral
proteins or peptides thereof to intestinal mucosa tissues or to cells and
monitoring chloride
secretion and/or intracellular calcium levels and/or cAMP levels.
Yet further, the present invention has also shown that administration of HIV
gp 160 to
6-7 day old Balb/C mice causes diarrhea in 100% of the cases.
It is another embodiment of the present invention to use the NSP4 protein or
peptides
thereof, including but not limited to NSP4 114-135, NSP4 120-147, NSP4 112-175
or NSP4
112-175 to identify and/or characterize a new intestinal receptor whose
signaling induces
secretion.
It is another embodiment of the present invention to provide a method for the
intentional induction of intestinal secretion including administration of the
NSP4 protein or
peptides thereof, including but not limited to NSP4 114-135, NSP4 120-14, NSP4
112-175 or
112-150.
It is another embodiment of the present invention to provide a method for the
treatment of cystic fibrosis including administration of NSP4 protein or
peptides thereof,
including but not limited to NSP4 114-135, NSP4 120-147, NSP4 112-175 or NSP4
112-150,
to enhance fluid secretion.
It is another embodiment of the present invention to provide a method for the
treatment of cystic fibrosis comprising administering NSP4 or derivatives or
new molecules
that act like NSP4 to enhance secretion through the same mechanism NSP4 uses.
It is another embodiment of the present invention to provide a new laxative
including
the NSP4 protein or peptides thereof, including but not limited to NSP4 114-
135, NSP4 120-
147, NSP4 112-175 or NSP4 112-150.
22
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
It is another embodiment of the present invention to provide methods for the
identification and use of compounds, such as small molecule inhibitors, to
bind the active
domain of NSP4, NSP4 peptides or fragments thereof (i.e., NSP4 114-135, NSP4
120-174,
NSP4 112-17S or NSP4 112-1S0) or other viral enterotoxins to prevent,
ameliorate or stop
S diarrheal disease. For the purpose of this invention, small molecule
inhibitors shall mean any
ligand that can bind with high affiuty to a target molecule, thereby
inhibiting the target
molecule's activity. Small molecule inhibitors include, but are not limited
to, peptides,
oligonucleotides, amino acids, derivatized amino acids, carbohydrates, and
organic and
inorganic chemicals. Libraries of small molecule inhibitors are available to
the practitioner
either according to known methods, or commercially. Accordingly, this method
includes
identifying a viral enterotoxin, screenng the purified enterotoxin against one
or more random
small molecule libraries, for example, a random peptide library, a random
oligonucleotide
library, or a pharmaceutical drug library, and identifying those small
molecules that bind with
high affinity to the viral enterotoxin.
1S As used herein the term "candidate substance" refers to any molecule that
may
potentially inhibit or enhance NSP4 or any fragment of NSP4 (i.e., NSP4 114-
135, NSP4
120-147, NSP4 112-17S or NSP4 112-150) activity. The candidate substance may
be a
protein or fragment thereof, a small molecule, or even a nucleic acid
molecule. It rnay prove
to be the case that the most useful pharmacological compounds will be
compounds that are
structurally related to NSP4 or any fragment of NSP4 (i.e., NSP4 114-135, NSP4
120-147,
NSP4 112-17S or NSP4 112-1S0). Using lead compounds to help develop improved
compounds is know as "rational drug design" and includes not only comparisons
with know
inhibitors and activators, but predictions relating to the structure of target
molecules.
The goal of rational drug design is to produce structural analogs of
biologically active
2S polypeptides or target compounds. By creating such analogs, it is possible
to fashion drugs,
which are more active or stable than the natural molecules, which have
different
susceptibility to alteration or which may affect the function of various other
molecules. In
one approach, one would generate a three-dimensional structure for a target
molecule, or a
fragment thereof. This could be accomplished by x-ray crystallography,
computer modeling
or by a combination of both approaches.
It also is possible to use antibodies to ascertain the structure of a target
compound
activator or inhibitor. In principle, this approach yields a pharmacore upon
which subsequent
23
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
drug design can be based. It is possible to bypass protein crystallography
altogether by
generating anti-idiotypic antibodies to a functional, pharmacologically active
antibody. As a
mirror image of a mirror image, the binding site of anti-idiotype would be
expected to be an
analog of the original antigen. The anti-idiotype could then be used to
identify and isolate
peptides from banks of chemically- or biologically-produced peptides. Selected
peptides
would then serve as the pharmacore. Anti-idiotypes may be generated using the
methods
described herein for producing antibodies, using an antibody as the antigen.
On the other hand, one may simply acquire, from various commercial sources,
small
molecule libraries that are believed to meet the basic criteria for useful
drugs in an effort to
"brute force" the identification of useful compounds. Screening of such
libraries, including
combinatorially generated libraries (e.g., peptide libraries), is a rapid and
efficient way to
screen large numbers of related (and unrelated) compounds for activity.
Combinatorial
approaches also lend themselves to rapid evolution of potential drugs by the
creation of
second, third and fourth generation compounds modeled of active, but otherwise
undesirable
compomzds.
Candidate compounds may include fragments or parts of naturally-occurring
compounds, or may be found as active combinations of known compounds, which
are
otherwise inactive. It is proposed that compounds isolated from natural
sources, such as
animals, bacteria, fungi, plant sources, including leaves and bark, and marine
samples may be
assayed as candidates for the presence of potentially useful pharmaceutical
agents. It will be
understood that the pharmaceutical agents to be screened could also be derived
or synthesized
from chemical compositions or man-made compounds. Thus, it is understood that
the
candidate substance identified by the present invention may be peptide,
polypeptide,
polynucleotide, small molecule inlubitors or ably other compounds that may be
designed
through rational drug design starting from known inhibitors or stimulators.
Other suitable modulators include antisense molecules, ribozymes, and
antibodies
(including single chain antibodies), each of wluch would be specific for the
target molecule.
Such compounds are described in greater detail elsewhere in this document. For
example, an
antisense molecule that bound to a translational or transcriptional start
site, or splice
junctions, would be ideal candidate inhibitors.
24
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
In addition to the modulating compounds initially identified, the inventors
also
contemplate that other sterically similar compounds may be formulated to mimic
the key
portions of the structure of the modulators. Such compounds, which may include
peptidomimetics of peptide modulators, may be used in the same manner as the
initial
modulators.
An inhibitor according to the present invention may be one which exerts its
inhibitory
or activating effect upstream, downstream or directly on NSP4 or any fragment
of NSP4 (i.e.,
NSP4 1 I4-135, NSP4 120-147, NSP4 112-175 or NSP4 112-150). Regardless of the
type of
inhibitor or activator identified by the present screening methods, the effect
of the inhibition
or activator by such a compound results in NSP4 or any fragment of NSP4 (i.e.,
NSP4 114-
135, NSP4 120-147, NSP4 112-175 or NSP4 112-150) as compared to that observed
in the
absence of the added candidate substance.
A quick, inexpensive and easy assay to run is an ira vitYO assay. Such assays
generally
use isolated molecules, can be run quickly and in large numbers, thereby
increasing the
amount of information obtainable in a short period of time. A variety of
vessels may be used
to run the assays, including test tubes, plates, dishes and other surfaces
such as dipsticks or
beads.
One example of a cell free assay is a binding assay. While not directly
addressing
function, the ability of a modulator to bind to a target molecule in a
specific fashion is strong
evidence of a related biological effect. For example, binding of a molecule to
a target may, in
and of itself, be inhibitory, due to steric, allosteric or charge-charge
interactions. The target
may be either free in solution, fixed to a support, expressed in or on the
surface of a cell.
Either the target or the compound may be labeled, thereby permitting
determination of
binding. ' Usually, the target will be the labeled species, decreasing the
chance that the
labeling will interfere with or enhance binding. Competitive binding formats
can be
performed in which one of the agents is labeled, and one may measure the
amount of free
label versus bound label to determine the effect on binding.
A technique for high throughput screening of compounds is described in WO
$4/03564. Large numbers of small peptide test compounds are synthesized on a
solid
substrate, such as plastic pins or some other surface. Bound polypeptide is
detected by
various methods.
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
The present invention also contemplates the screening of compounds for their
ability
to modulate NSP4 or any fragment of NSP4 (i.e., NSP4 114-135, NSP4 120-147,
NSP4 112-
175 or NSP4 112-150) in cells. Various cell lines can be utilized for such
screening assays,
including cells specifically engineered for this purpose.
Depending on the assay, culture may be required. The cell is examined using
any of a
number of different physiologic assays. Alternatively, molecular analysis may
be performed,
for example, protein expression, mRNA expression (including differential
display of whole
cell or polyA RNA) and others.
In vivo assays involve the use of various animal models, including transgenic
animals
that have been engineered to have specific defects, or carry markers that can
be used to
measure the ability of a candidate substance to reach and affect different
cells within the
organism. Due to their size, ease of handling, and information on their
physiology and
genetic make-up, mice are a preferred embodiment, especially for transgenics.
However,
other animals are suitable as well, including rats, rabbits, hamsters, guinea
pigs, gerbils,
woodchucks, cats, dogs, sheep, goats, pigs, cows, horses and monkeys
(including chimps,
gibbons and baboons). Assays for modulators may be conducted using an animal
model
derived from any of these species.
In such assays, one or more candidate substances are administered to an
animal, and
the ability of the candidate substances) to alter one or more characteristics,
as compared to a
similar animal not treated with the candidate substance(s), identifies a
modulator. The
characteristics may be any of those discussed above with regard to the
function of a particular
compound or cell, or instead a broader indication such as behavior, anemia,
immune
response, etc.
Treatment of these animals with test compounds will involve the administration
of the
compound, in an appropriate form, to the animal. Administration will be by any
route that
could be utilized for clinical or non-clinical purposes, including but not
limited to oral,
intranasal, buccal, or even topical. Alternatively, administration may be by a
parenteral
route, e.g intradermal, subcutaneous, intramuscular, intraperitoneal or
intravenous injection.
Determining the effectiveness of a compound in vivo may involve a variety of
different criteria. Also, measuring toxicity and dose response can be
performed in animals in
a more meaningful fashion than in ifa vity~o or irZ cyto assays.
26
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
Another method for identifying small molecule inhibitors includes the steps of
identifying viral enterotoxins, determining the high resolution structure of
these proteins
and/or peptides thereof, determinng the active domains) and designing small
molecule
inhibitors which bind with high affinity to the active domain(s). Another
method includes
identifying viral enterotoxins, identifying the intestinal receptor which
binds the viral
enterotoxin, and designing small molecule iWibitors which competitively bind
the receptor,
without inducing secretion.
It is another embodiment of the present invention to provide a method for the
desig~i
of new drugs for the prevention of diarrhea and/or Ca2+ mediated intestinal
secretion
including identifying the intracellular pathway by which [Ca2+]; is increased
or downstream
between Caz+ mobilization and chloride channel activation or identifying
channel blocking
and making compounds which inhibit any step in the pathways. Specifically,
this method
includes identifying the molecules active in the signaling pathway and
identifying
compounds which inhibit their activity. Such compounds will include but not be
limited to
small molecule inhibitors which bloclc binding of NSP4 to its receptor,
blocking of G protein
mediated or other signal transduction secondary messengers and pathways which
lead to
chloride secretion or diarrhea.
It is another embodiment of the present invention to provide a method for the
diagnosis of rotavinus infection including the detection of NSP4 in stools of
individuals with
diarrhea. Detection of peptides of NSP4 is considered to fall within the scope
of detection of
NSP4.
It is another embodiment of the present invention to provide a method for the
diagnosis of rotavirus infection including the detection of antibodies to NSP4
in the sera or
stools of individuals with diarrhea.
It is another embodiment of the present invention to provide a vaccine
comprising the
NSP4 protein or peptides thereof, including but not limited to NSP4 114-135,
NSP4 120-147,
NSP4 112-175 or NSP4 112-150 to induce the formation of protective active or
passive
antibodies.
It is another embodiment of the present invention to provide a vaccine
comprising a
toxoid form of the NSP4 protein or fragments thereof, including but not
limited to
formaldehyde, heat inactivated or mutated NSP4 to induce the formation of a
protective
27
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
immune response. Fragments of NSP4 include, but are not limited to, NSP4 114-
135, NSP4
120-147, NSP4 112-175 or NSP4 112-150.
It is another embodiment of the present invention to provide a method to
monitor
vaccine efficacy or protective immzuuty by determining the immune response to
NSP4
protein and/or to peptides thereof.
It is another embodiment of the present invention to provide a method for
immunization against rotavirus infection comprising administering to a subject
a vaccine
including the NSP4 protein or peptides thereof, including, but not limiting
to, NSP4 114-135,
NSP4 120-147, NSP4 112-175 or NSP4 112-150 peptides.
It is another embodiment of the present invention to provide a method of
passive
immunization against rotavirus infection including administering to an
expectant mother a
vaccine including the NSP4 protein or peptides thereof, including but not
limiting to, NSP4
114-135, NSP4 120-147, NSP4 112-175 or NSP4 112-150.
It is another embodiment of the present invention to provide a method for
immunization against rotavirus infection comprising administering to a subj
ect a vaccine
comprising a toxoid form of the NSP4 protein.
It is another embodiment of the present invention to provide a method for
immuuzation against rotavirus infection comprising administering to a subject
a vaccine
comprising a non-glycosylated NSP4 protein.
For an antigenic composition to be useful as a vaccine, an antigenic
composition must
induce an immune response to the antigen in a cell, tissue or animal (e.g., a
human). As used
herein, an "antigenic composition" may comprise an antigen (e.g., a NSP4
peptide or NSP4
polypeptide or NSP4 protein or toxoid thereof), a nucleic acid encoding an
antigen (e.g., an
antigen expression vectox), or a cell expressing or presenting an antigen. In
particular
embodiments the antigenic composition comprises the nucleic acid sequence that
encodes
NSP4, or any fragments thereof, including but not limiting to NSP4 112-135,
NSP4 120-147,
NSP4 112-175 or NSP4 112-150, or an immunologically functional equivalent
thereof. In
other embodiments, the antigenic composition is in a mixture that comprises an
additional
ixnmunostimulatory agent or nucleic acids encoding such an agent.
Immunostimulatory
agents include but are not limited to an additional antigen, an
immunomodulator, an antigen
28
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
presenting cell or an adjuvant. In other embodiments, one or more of the
additional agents)
is covalently bonded to the antigen or an immunostimulatory agent, in any
combination. In
certain embodiments, the antigenc composition is conjugated to or comprises an
HLA
anchor motif amino acids.
In certain embodiments, an antigenic composition or immunologically functional
equivalent, may be used as an effective vaccine in inducing a humoral and/or
cell-mediated
immune response in an animal. The present invention contemplates one or more
antigenic
compositions or vaccines for use in both active and passive immunization
embodiments.
A vaccine of the present invention may vary in its composition of
proteinaceous,
nucleic acid and/or cellular components. In a non-limiting example, a nucleic
encoding an
antigen might also be formulated with a proteinaceous adjuvant. Of course, it
will be
understood that various compositions described herein may further comprise
additional
components. For example, one or more vaccine components may be comprised in a
lipid or
liposome. In another non-limiting example, a vaccine may comprise one or more
adjuvants.
A vaccine of the present invention, and its various components, may be
prepared and/or
admiustered by any method disclosed herein or as would be known to one of
ordinary skill in
the art, in light of the present disclosure.
It is understood that an antigenic composition of the present invention may be
made
by a method that is well knovm in the art, including but not limited to
chemical synthesis by
solid phase synthesis and purification away from the other products of the
chemical reactions
by HPLC, or production by the expression of a nucleic acid sequence encoding a
peptide or
polypeptide comprising an antigen of the present invention in an in vity-o
translation system or
in a living cell. Preferably the antigenic composition isolated and
extensively dialyzed to
remove one or more undesired small molecular weight molecules and/or
lyophilized for more
ready formulation into a desired vehicle. It is further understood that
additional amino acids,
mutations, chemical modification and such like, if any, that are made in a
vaccine component
will preferably not substantially interfere with the antibody recognition of
the epitopic
sequence.
A peptide or polypeptide corresponding to one or more antigenic determinants
of the
present invention should generally be at least five or six amino acid residues
in length, and
may contain up to about 10, about 15, about 20, about 25 about 30 ,about 35,
about 40, about
29
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
45 or about 50 residues or so. A peptide sequence may be synthesized by
methods known to
those of ordinary skill in the art, such as, for example, peptide synthesis
using automated
peptide synthesis machines, such as those available from Applied Biosystems
(Foster City,
CA).
Longer peptides or polypeptides also may be prepared, e.g., by recombinant
means.
In certain embodiments, a nucleic acid encoding an antigenic composition
and/or a
component described herein may be used, for example, to produce an ant~genc
composition
in vitro or in vivo for the various compositions and methods of the present
invention. For
example, in certain embodiments, a nucleic acid encoding an antigen is
comprised in, for
example, a vector in a recombinant cell. The nucleic acid may be expressed to
produce a
peptide or polypeptide comprising an antigenic sequence. The peptide or
polypeptide may be
secreted from the cell, or comprised as part of or within the cell.
In certain embodiments, an immune response may be promoted by transfecting or
inoculating an animal with a nucleic acid encoding an antigen. One or more
cells comprised
within a target animal then expresses the sequences encoded by the nucleic
acid after
administration of the nucleic acid to the animal. Thus, the vaccine may
comprise "genetic
vaccine" useful for immunization protocols. A vaccine may also be in the form,
for example,
of a nucleic acid (e.g., a cDNA or an RNA) encoding all or part of the peptide
or polypeptide
sequence of an antigen. Expression ih vivo by the nucleic acid may be, for
example, by a
plasmid type vector, a viral vector, or a viral/plasmid construct vector.
In other aspects, the nucleic acid comprises a coding region that encodes all
or part of
the NSP4, NSP4 114-135, NSP4 120-147, NSP4 112-175 or NSP4 112-150, or an
immunologically functional equivalent thereof. Of course, the nucleic acid may
comprise
and/or encode additional sequences, including but not limited to those
comprising one or
2S more immunomodulators or adjuvants. The nucleotide and protein, polypeptide
and peptide
' encoding sequences for various genes have been previously disclosed, and may
be found at
computerized databases known to those of ordinary shill in the art. One such
database is the
National Center for Biotechnology Information's Genbank and GenPept databases
(http://www.ncbi.nlm.nih.gov/). The coding regions for these known genes may
be
amplified, combined with the sequences NSP4 (SEQ.ID.N0:16) , NSP4 114-135,
NSP4 120-
147, NSP4 112-175 or NSP4 112-150 disclosed herein (e.g., ligated) and/or
expressed using
the techniques disclosed herein or by any technique that would be l~now to
those of ordinary
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
skill in the art (e.g., Sambrook et al., 1987). Though a nucleic acid may be
expressed in an
ifz vitf°o expression system, in preferred embodiments the nucleic acid
comprises a vector for
in vivo replication and/or expression.
Specifically, nucleic acids according to the present invention may encode an
entire
NSP4 gene, a domain of NSP4, or any other fragment of NSP4 as set forth
herein. The
nucleic acid may be derived from genomic DNA, i.e., cloned directly from the
genome of a
particular organism. The following sequences are sequences corresponding to
NSP4 genes
and are within the scope of the invention and are referenced with the
corresponding GenBank
Accession Numbers http://www.ncbi.nlm.nih.gov/Genbank/GenbankSearch.html):
ALA(SEQ.ID.N0:17, AF144792); C-11 (SEQ.ID.NO:18, AF144793); R-2 (SEQ.ID.N0:19,
AF 144794); BAP-2 (SEQ.ID.N0:20, AF 144795); BAPwt (SEQ.ID.N0:21, AF 144796);,
A253 (SEQ.ID.N0:22, AF144797); A131 (SEQ.ID.N0:23, AF144798); A411
(SEQ.ID.N0:24, AF144799); A34 (SEQ.ID.NO:25, AF165219); H-2 (SEQ.ID.NO:26,
AF144801); FI-23 (SEQ.ID.N0:27, AF144802); FI-14 (SEQ.ID.N0:28, AF144803);
BRV033 (SEQ.ID.N0:29, AF144804); B223 (SEQ.ID.N0:30, AF144805); CU-1
(SEQ.ID.N0:31, AF144806); OSU (SEQ.ID.N0:32, D88831); and SAll (SEQ.ID.N0:33,
AF087678). Also included in the scope of the present invention is the nucleic
acid sequences
for other rotavirus genes including, but not limiting to : VP6(SEQ.ID.N0:34,
D00325);
VP6(SEQ.ID.N0:35, K02086) and VP2(SEQ.ID.N0:36, X14949).
It is also contemplated that an antigenic composition of the invention may be
combined with one or more additional components to form a more effective
vaccine. Non-
limiting examples of additional components include, for example, one or more
additional
antigens, immunomodulators or adjuvants to stimulate an immune response to an
antigenic
composition of the present invention and/or the additional component(s).
In particular embodiments, it is contemplated that nucleic acids encoding
antigens of
the present invention may be transfected into plants, particularly edible
plants, and all or part
of the plant material used to prepare a vaccine, such as for example, an oral
vaccine. Such
methods are described in U.S. Patent Nos. 5,484,719, 5,612,487, 5,914,123,
5977,438 and
6,034,298, each incorporated herein by reference.
For example, it is contemplated that immunomodulators can be included in the
vaccine to augment a cell's or a patient's (e.g., an animal's) response.
Immunomodulators
31
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
can be included as purif ed proteins, nucleic acids encoding immunomodulators,
and/or cells
that express immunomodulators in the vaccine composition. The following
sections list non
limiting examples of imznunomodulators that are of interest, and it is
contemplated that
various combinations of immunomodulators may be used in certain embodiments
(e.g., a
cytokine and ~a chemokine).
Interleukins, cytokines, nucleic acids encoding interleukins or cytokines,
and/or cells
expressing such compounds are contemplated as possible vaccine components.
Interleukins
and cytokines, include but are not limited to interleukin 1 (IL-1), IL-2, IL-
3, IL-4, IL-5, IL-6,
IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-18, [3-
interferon, a-interferon,
y-interferon, angiostatin, thrombospondin, endostatin, GM-CSF, G-CSF, M-CSF,
METH-l,
METH-2, tumor necrosis factor, TGF(3, LT and combinations thereof.
Chemolcines, nucleic acids that encode for chemokines, and/or cells that
express such
also may be used as vaccine components. Chemokines generally act as
chemoattractants to
recruit immune effector cells to the site of chemolcine expression. It may be
advantageous to
express a particular chemokine coding sequence in combination with, for
example, a cytokine
coding sequence, to enhance the recruitment of other immune system components
to the site
of treatment. Such chemokines include, fox example, RANTES, MCAF, MIP1-alpha,
MIP1
Beta, IP-10 and combinations thereof. The skilled artisan will recognize that
certain
cytol~ines are also known to have chemoattractant effects and could also be
classified under
the term chemokines.
In certain embodiments, an antigenic composition's may be chemically coupled
to a
carrier or recombinantly expressed with a immunogenic carrier peptide or
polypeptide (e.g., a
antigen-carrier fusion peptide or polypeptide) to enhance an immune reaction.
Exemplary
and preferred immunogenic carrier amino acid sequences include hepatitis B
surface antigen,
keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins
such
as ovalbumin, mouse serum albumin or rabbit serum albumin also can be used as
immunogenic Garner proteins. Means for conjugating a polypeptide or peptide to
an
immunogenic carrier protein are well known in the art and include, for
example,
glutaraldehyde, m-maleimidobenzoyl-N-hydroxysuccinimide ester, carbodiimide
and bis
biazotized benzidine.
32
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
It may be desirable to co-administer biologic response modifiers (BRM), which
have
been shown to upregulate T cell immunity or downregulate suppressor cell
activity. Such
BRMs include, but are not limited to, cimetidine (CIM; 1200 mg/d)
(Smith/I~line, PA); low-
dose cyclophosphamide (CYP; 300 mg/m2) (Johnson/ Mead, NJ), or a gene encoding
a
protein involved in one or more immune helper functions, such as B-7.
T_mmunization protocols have used adjuvants to stimulate responses for many
years,
and as such adjuvants are well known to one of ordinary skill in the art. Some
adjuvants
affect the way in which antigens are presented. For example, the immune
response is
increased when protein antigens are precipitated by alum. Emulsification of
antigens also
prolongs the duration of antigen presentation.
In certain embodiments, an adjuvant effect is achieved by use of an agent such
as
alum used in about 0.05 to about 0.1% solution in phosphate buffered saline.
Alternatively,
the antigen is made as an admixture with synthetic polymers of sugars
(Carbopol0) used as
an about 0.25% solution. Adjuvant effect may also be made by aggregation of
the antigen in
the vaccine by heat treatment with temperatures ranging between about
70° to about 101°C
for a 30-second to 2-minute period, respectively.
Some adjuvants, for example, are certain organic molecules obtained from
bacteria,
act on the host rather than on the antigen. An example is muramyl dipeptide (N-
acetylmuramyl-L-alanyl-D-isoglutamine [MDP]), a bacterial peptidoglycan. The
effects of
MDP, as with most adjuvants, are not fully understood. MDP stimulates
macrophages but
also appears to stimulate B cells directly. The effects of adjuvants,
therefore, are not antigen-
specific. If they are administered together with a purified antigen, however,
they can be used
to selectively promote the response to the antigen.
Adjuvants have been used experimentally to promote a generalized increase in
immunity against unlmown antigens (e.g., U.S. Patent 4,877,611).
In certain embodiments, hemocyanins and hemoerythrins may also be used in the
invention. The use of hemocyanin from keyhole limpet (KLH) is preferred in
certain
embodiments, although other molluscan and arthropod hemocyanins and
hemoerythrins may
be employed.
33
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
Various polysaccharide adjuvants may also be used. For example, the use of
various
pneumococcal polysaccharide adjuvants on the antibody responses of mice has
been
described (Yin et al., 1989). The doses that produce optimal responses, or
that otherwise do
not produce suppression, should be employed as indicated (Yin et al., 1989).
Polyamine
varieties of polysaccharides are particularly preferred, such as chitin and
chitosan, including
deacetylated chitin.
Another group of adjuvants are the muramyl dipeptide (MDP, N-acetylmuramyl-L-
alanyl-D-isoglutamine) group of bacterial peptidoglycans. Derivatives of
muramyl dipeptide,
such as the amino acid derivative threonyl-MDP, and the fatty acid derivative
MTPPE, are
also contemplated.
U.S. Patent 4,950,645 describes a lipoplulic disaccharide-tripeptide
derivative of
muramyl dipeptide which is described for use in artificial liposomes formed
from
phosphatidyl choline and phosphatidyl glycerol. It is the to be effective in
activating human
monocytes and destroying tumor cells, but is non-toxic in generally high
doses. The
compounds of U.S. Patent 4,950,645 and PCT Patent Application WO 91/16347, are
contemplated for use with cellular carriers and other embodiments of the
present invention.
Another adjuvant contemplated for use in the present invention is BCG. BCG
(bacillus Calmette-Guerin, an attenuated strain of Mycobacterium) and BCG-cell
wall
skeleton (CWS) may also be used as adjuvants in the invention, with or without
trehalose
dimycolate. Trehalose dimycolate may be used itself. Trehalose dimycolate
administration
has been shown to correlate with augmented resistance to influenza virus
infection in mice
(Azuma et al., 1988). Trehalose dimycolate may be prepared as described in
U.S. Patent
4,579,945.
BCG is an important clinical tool because of its immunostimulatory properties.
BCG
acts to stimulate the reticulo-endothelial system, activates natural killer
cells and increases
proliferation of hematopoietic stem cells. Cell wall extracts of BCG have
proven to have
excellent immune adjuvant activity. Molecular genetic tools and methods for
mycobacteria
have provided the means to introduce foreign genes into BCG (Jacobs et al.,
1987; Snapper et
al., 1988; Husson et al., 1990; Martin et al., 1990).
Live BCG is an effective and safe vaccine used worldwide to prevent
tuberculosis.
BCG and other mycobacteria are highly effective adjuvants, and the immune
response to
34
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
mycobacteria has been studied extensively. With nearly 2 billion
immunizations, BCG has a
long record of safe use in man (Luelmo 1982; Lotte et al., 1984). It is one of
the few
vaccines that can be given at birth, it engenders long-lived immune responses
with only a
single dose, and there is a worldwide distribution network with experience in
BCG
vaccination. An exemplary BCG vaccine is sold as TICE~ BCG (Organon Inc., West
Orange, NJ)'.
In a typical practice of the present invention, cells of Mycobacterium bovis-
BCG are
grown and harvested by methods lffzown in the art. For example, they may be
grown as a
surface pellicle on a Sauton medium or in a fermentation vessel containing the
dispersed
culture in a Dubos medium (Dubos et al., 1947; Rosenthal 1937). All the
cultures are
harvested after 14 days incubation at about 37°C. Cells grown as a
pellicle are harvested by
using a platinum loop whereas those from the fermenter are harvested by
centrifugation or
tangential-flow filtration. The harvested cells are resuspended in an aqueous
sterile buffer
medium. A typical suspension contains from about 2xI01° cells/ml to
about 2xI012 cellslml.
To this bacterial suspension, a sterile solution containing a selected enzyme
which will
degrade the BCG cell covering material is added. The resultant suspension is
agitated such as
by stirring to ensure maximal dispersal of the BCG organisms. Thereafter, a
more
concentrated cell suspension is prepared and the enzyme in the concentrate
removed,
typically by washing with an aqueous buffer, employing known techniques such
as
tangential-flow filtration. The enzyme-free cells are adjusted to an optimal
immunological
concentration with a cryoprotectant solution, after which they are filled into
vials, ampoules,
etc., and lyophilized, yielding BCG vaccine, which upon reconstitution with
water is ready
for immunization.
Amphipathic and surface active agents, e.g., saponin and derivatives such as
QS21
(Cambridge Biotech), form yet another group of adjuvants for use with the
immunogens of
the present invention. Nonionic block copolymer surfactants (Rabinovich et
al., 1994;
Hunter et al., 1991) may also be employed. Oligonucleotides axe another useful
group of
adjuvants (Yamamoto et al., 1988). Quil A and lentinen are other adjuvants
that may be used
in certain embodiments of the present invention.
One group of adjuvants also contemplated for use in the invention are the
detoxified
endotoxins, such as the refined detoxified endotoxin of U.S. Patent 4,866,034.
These refined
detoxified endotoxins are effective in producing adjuvant responses in
mammals. The
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
combination of detoxified endotoxins with trehahose dimycolate is also
contemplated, as
described in U.S. Patent 4,435,386. Combinations of detoxified endotoxins with
trehalose
dimycolate and endotoxic glycolipids is also contemplated (U.S. Patent
4,SOS,899), as is
combination of detoxified endotoxins with cell wall skeleton (CWS) or CWS and
trehalose
S dimycolate, as described in U.S. Patents 4,436,727, 4,436,728 and 4,SOS,900.
Combinations
of just CWS and trehahose dimycolate, without detoxified endotoxins, is also
envisioned to be
useful, as described in U.S. Patent 4,520,019.
One skilled in the art is cognizant that the NSP4 peptide (NSP4 114-135, NSP4
120-
147, NSP4 112-17S or NSP4 112-1S0) or toxoid thereof may be produced
synthetically or by
an expression vector. Expression vectors that may be used include, but are not
limited to
mammalian, yeast, viral, bacterial, plant or insect.
Another embodiment of the present invention is a fusion protein. One of skill
in the
art is cognizant that fusion proteins are generated using standard molecular
biology
techniques well known in the art. Further, one of skill in the art is aware
that in the present
1 S invention the term "linked" can be used interchangeably with the term
"fused". The fusion
protein comprises a NSP4 peptide linked to a protein that forms a virus-like
particle. The
NSP4 peptide may include, but is not limited to, NSP4 114-135, NSP4 120-147,
NSP4 112-
175 or NSP4 112-150. The virus-like particle is a viral protein or peptide
isolated from
Caliciviridae or Reoviridae. Specifically, the viral protein or peptide
isolated from
Caliciviy°idae is a Norwalk virus protein or peptide and the viral
protein or peptide isolated
from Reoviridae is a rotavirus protein or peptide. The Norwalk virus protein
or peptide may
be ORFZ or ORF3 or ORF2 plus ORF3 or a toxoid thereof. The rotavirus protein
or peptide
may be VP2, VP4, VPS, VP6 and VP7. In specific embodiments, the rotavirus
peptide is
VP2.
2S It is well known that virus-lihce particles (VLPs) consist of capsid
proteins assembled
into a shell-like structure without the presence of viral nucleic acid within
the shell. These
shells can display conformational epitopes that are not present on individual
capsid proteins.
The use of VLPs offer several immunogenic advantages. First, VLPs present
conformational
epitopes to the immune system in such a way as native infectious particles so
that
neutralizing antibodies and other protective immune responses are induced
effectively.
Second, because VLPs are noninfectious, inactivation is not required. Thus,
one skilled in
the art realizes that the use of virus-like particles may be better immunogens
than formahin-
36
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
inactivated whole-virus vaccines or proteins. Thus, it can be appreciated that
the fusion
protein of the present invention may be a better immunogen than the NSP4
alone.
Another embodiment of the present invention comprises an expression vector
comprising a nucleic acid sequence encoding a fusion protein, wherein the
fusion protein
comprises a NSP4 peptide and a viral peptide that forms a virus-life particle.
The nucleic
acid sequence is operatively linked to a promoter sequence.
As used herein the term "vector" is used to refer to a carrier nucleic acid
molecule
into which a nucleic acid sequence can be inserted for introduction into a
cell where it can be
replicated. A nucleic acid sequence can be "exogenous," which means that it is
foreign to the
cell into which the vector is being introduced or that the sequence is
homologous to a
sequence in the cell but in a position within the host cell nucleic acid in
which the sequence is
ordinarily not found. Vectors include plasmids, cosmids, viruses
(bacteriophage, animal
viruses, and plant viruses), and artificial chromosomes (e.g., YACs). One of
shill in the art
would be well equipped to construct a vector through standard recombinant
techniques,
which are described in Maniatis et al., 1988 and Ausubel et al., 1994, both
incorporated
herein by reference.
The term "expression vector" refers to a vector containing a nucleic acid
sequence
coding for at least part of a gene product capable of being transcribed. In
some cases, RNA
molecules are then translated into a protein, polypeptide, or peptide. In
other cases, these
sequences are not translated, for example, in the production of antisense
molecules or
ribozymes. Expression vectors can contain a variety of "control sequences,"
which refer to
nucleic acid sequences necessary for the transcription and possibly
translation of an operably
linked coding sequence in a particular host organism. In addition to control
sequences that
govern transcription and hanslation, vectors and expression vectors may
contain nucleic acid
sequences that serve other functions as well and are described infra.
A "promoter" is a control sequence that is a region of a nucleic acid sequence
at
which initiation and rate of transcription are controlled. It may contain
genetic elements at
which regulatory proteins and molecules may bind such as RNA polymerase and
other
transcription factors. The phrases "operatively positioned," "operatively
linlced," "tinder
control," and "under transcriptional control" mean that a promoter is in a
correct functional
location andlor orientation in relation to a nucleic acid sequence to control
transcriptional
37
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
initiation and/or expression of that sequence. A promoter may or may not be
used in
conjunction with an "enhancer," which refers to a cis-acting regulatory
sequence involved in
the transcriptional activation of a nucleic acid sequence.
A promoter may be one naturally associated with a gene or sequence, as may be
obtained by isolating the 5' non-coding sequences located upstream of the
coding segment
and/or exon. Such a promoter can be referred to as "endogenous." Similarly, an
enhancer
may be one naturally associated with a nucleic acid sequence, located either
downstream or
upstream of that sequence. Alternatively, certain advantages will be gained by
positioning
the coding nucleic acid segment under the control of a recombinant or
heterologous promoter,
which refers to a promoter that is not normally associated with a nucleic acid
sequence in its
natural environment. A recombinant or heterologous enhancer refers also to an
enhancer not
normally associated with a nucleic acid sequence in its natural enviromnent.
Such promoters
or enhancers may include promoters or enhancers of other genes, and promoters
or enhancers
isolated from any other prokaryotic, viral, or eulcaryotic cell, and promoters
or enhancers not
"naturally occurring," i.e., containing different elements of different
transcriptional
regulatory regions, and/or mutations that alter expression. In addition to
producing nucleic
acid sequences of promoters and enhancers synthetically, sequences may be
produced using
recombinant cloning and/or nucleic acid amplification technology, including
PCRTM, in
connection with the compositions disclosed herein (see U.S. Patent 4,683,202,
U.S. Patent
5,928,906, each incorporated herein by reference). Furthermore, it is
contemplated the
control sequences that direct transcription and/or expression of sequences
within non-nuclear
organelles such as mitochondria, chloroplasts, and the like, can be employed
as well.
Naturally, it will be important to employ a promoter and/or enhancer that
effectively
directs the expression of the DNA segment in the cell type, organelle, and
organism chosen
for expression. Those of slcill in the art of molecular biology generally know
the use of
promoters, enhancers, and cell type combinations for protein expression, for
example, see
Sambrook et al. (1989), incorporated herein by reference. The promoters
employed may be
constitutive, tissue-specific, inducible, and/or useful under the appropriate
conditions to
direct high level expression of the intTOduced DNA segment, such as is
advantageous in the
large-scale production of recombinant proteins and/or peptides. The promoter
may be
heterologous or endogenous.
38
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
A specific initiation signal also may be required for efficient translation
of,coding
sequences. These signals include the ATG initiation codon or adjacent
sequences.
Exogenous translational control signals, including the ATG initiation codon,
may need to be
provided. One of ordinary slcill in the art would readily be capable of
determining this and
providing the necessary signals. It is well known that the initiation codon
must be "in-frame"
with the reading frame of the desired coding sequence to ensure translation of
the entire
insert. The exogenous translational control signals and initiation codons can
be either natural
or synthetic. The efficiency of expression may be enhanced by the inclusion of
appropriate
transcription enhancer elements.
Vectors can include a multiple cloning site (MCS), which is a nucleic acid
region that
contains multiple restriction enzyme sites, any of which can be used in
conjunction with
standard recombinant technology to digest the vector. (See Carbonelli et al.,
1999, Levenson
et al., 1998, and Cocea, 1997, incorporated herein by reference.) "Restriction
enzyme
digestion" refers to catalytic cleavage of a nucleic acid molecule with an
enzyme that
functions only at specific locations in a nucleic acid molecule. Many of these
restriction
enzymes are commercially available. Use of such enzymes is widely understood
by those of
skill in the art. Frequently, a vector is linearized or fragmented using a
restriction enzyne
that cuts within the MCS to enable exogenous sequences to be ligated to the
vector.
"Ligation" refers to the process of forming phosphodiester bonds between two
nucleic acid
fragments, which may or may not be contiguous with each other. Techniques
involving
restriction enzymes and ligation reactions are well known to those of skill in
the art of
recombinant technology.
In order to propagate a vector in a host cell, it may contain one or more
origins of
replication sites (often termed "ori"), which is a specific nucleic acid
sequence at which
replication is initiated. Alternatively an autonomously replicating sequence
(ARS) can be
employed if the host cell is yeast.
In certain embodiments of the invention, the cells contain nucleic acid
construct of the
present invention, a cell may be identified in vitro or ih. vivo by including
a marker in the
expression vector. Such markers would confer an identifiable change to the
cell permitting
easy identification of cells containing the expression vector. Generally, a
selectable marker is
one that confers a property that allows for selection. A positive selectable
marker is one in
which the presence of the marker allows for its selection, while a negative
selectable marker
39
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
is one in which its presence prevents its selection. An example of a positive
selectable
marker is a drug resistance marker.
Usually the inclusion of a drug selection marker aids in the cloning and
identification
of transformants, for example, genes that confer resistance to neomycin,
puromycin,
hygromycin, DHFR, GPT, zeocin and histidinol are useful selectable markers. In
addition to
markers conferring a phenotype that allows for the discrimination of
transformants based on
the implementation of conditions, other types of markers including screenable
markers such
as GFP, whose basis is colorimetric analysis, are also contemplated.
Alternatively,
screenable enzymes such as herpes simplex virus thymidine l~inase (tk) or
chloramphenicol
acetyltransferase (CAT) may be utilized. One of slcill in the art would also
know how to
employ immunologic markers, possibly in conjunction with FAGS analysis. The
marker used
is not believed to be important, so long as it is capable of being expressed
simultaneously
with the nucleic acid encoding a gene product. Further examples of selectable
and screenable
marlcers are well l~nown to one of skill in the art.
As used herein, the terms "cell," "cell line," and "cell culture" may be used
interchangeably. All of these terms also include their progeny, which is any
and all
subsequent generations. It is understood that all progeny may not be identical
due to
deliberate or inadvertent mutations. In the context of expressing a
heterologous nucleic acid
sequence, "host cell" refers to a prokaryotic or eukaryotic cell, and it
includes any
transformable organisms that is capable of replicating a vector and/or
expressing a
heterologous gene encoded by a vector. A host cell can, and has been, used as
a recipient for
vectors. A host cell may be "transfected" or "transformed," which refers to a
process by
which exogenous nucleic acid is transferred or introduced into the host cell.
A transformed
cell includes the primary subject cell and its progeny.
Host cells may be derived from prokaryotes or eulcaryotes, depending upon
whether
the desired result is replication of the vector or expression of part or all
of the vector-encoded
nucleic acid sequences. Numerous cell lines and cultures are available for use
as a host cell,
and they can be obtained through tk~e American Type Culture Collection (ATCC),
which is an
organization that serves as an archive for living cultures and genetic
materials
(www.atcc.org). An appropriate host can be determined by one of skill in the
art based on the
vector backbone and the desired result. A plasmid or cosmid, for example, can
be introduced
into a prokaryote host cell for replication of many vectors. Bacterial cells
used as host cells
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
for vector replication and/or expression include DHSa, JM109, and KCB, as well
as a number
of commercially available bacterial hosts such as SURE~ Competent Cells and
SOLOPACKTM Gold Cells (STRATAGENE~, La Jolla). Alternatively, bacterial cells
such
as E. coli LE392 could be used as host cells for phage viruses.
Examples of eukaryotic host cells for replication and/or expression of a
vector include
HeLa, NIH3T3,~Jurkat, 293, Cos, CHO, Saos, and PC12. Many host cells from
various cell
types and organisms are available and would be known to one of skill in the
art. Similarly, a
viral vector may be used in conjunction with either a eulcaryotic or
prokaryotic host cell,
particularly one that is permissive for replication or expression of the
vector.
Some vectors may employ control sequences that allow it to be replicated
and/or
expressed in both prokaryotic and eukaryotic cells. One of skill in the art
would further
understand the conditions under which to incubate all of the above described
host cells to
maintain them and to permit replication of a vector. Also understood and known
are
techniques and conditions that would allow large-scale production of vectors,
as well as
production of the nucleic acids encoded by vectors and their cognate
polypeptides, proteins,
or peptides.
Numerous expression systems exist that comprise at Ieast a part or all of the
compositions discussed above. Prokaryote- and/or eukaryote-based systems can
be employed
for use with the present invention to produce nucleic acid sequences, or their
cognate
polypeptides, proteins and peptides. Many such systems are commercially and
widely
available.
The insect celh/baculovirus system can produce a high level of protein
expression of a
heterologous nucleic acid segment, such as described in U.S. Patent No.
5,871;986,
4,879,236, both herein incorporated by reference, and which can be bought, for
example,
under the name MAXBACOO 2.0 from INVITROGEN~ and BACPACKTM BACULOVIRUS
EXPRESSION SYSTEM FROM CLONTECH~.
Other examples of expression systems include STRATAGENE~'s COMPLETE
CONTROLT"" Inducible Mammalian Expression System, which involves a synthetic
ecdysone-inducibhe receptor, or its pET Expression System, an E. coli
expression system.
Another example of an inducible expression system is available from
INVITROGEN~,
41
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
which carries the T-REXTM (tetracycline-regulated expression) System, an
inducible
mammalian expression system that uses the full-length CMV promoter.
INVITROGEN~
also provides a yeast expression system called the Pichia methanolica
Expression System,
which is designed for high-level production of recombinant proteins in the
methylotrophic
yeast Pichia methanolica. One of skill in the art would know how to express a
vector, such as
an expression construct, to produce a nucleic acid sequence or its cognate
polypeptide,
protein, or peptide.
It is another embodiment of the present invention to provide vaccines against
gastroenteritis viruses, including rotaviruses, caliciviruses, astroviruses,
enteric adenoviruses,
coronoviruses and parvoviruses, including viral enterotoxins which induce the
diarrhea
associated with viral infection.
It is another embodiment of the present invention to provide methods for the
identification of potential vaccines against gastroenteritis, viruses,
including screening for
viral enterotoxins, raising antibodies against any identified possible
enterotoxins, and
determining whether the antibodies protect against disease caused by the
virus.
It is another embodiment of the present invention to provide a method of
identifying a
virulent strain of rotavirus by determining the amino acid sequence of the
NSP4 protein of
the strain.
It is another embodiment of the present invention to identify key residues in
NSP4
responsible for its ability to induce diarrhea and thus to identify specific
amino acid
sequences associated with avirulence. Having done this, gene 10 from these
avirulent viruses
may be used to select and produce reassortment viruses that contain a gene 10
that confers an
avirulent phenotype which can be used as live attenuated reassortment virus
vaccine
candidates.
It is another embodiment of the present invention to provide methods for the
screening of antiviral compounds, compositions and/or treatments and/or the
evaluation of
vaccine efficacy, including administering viruses, viral proteins or peptides
thereof to one or
more of three new animal models for diarrheal virus infections, the CDl mouse,
the Balb/C
mouse and the Sprague-Dawley rat.
42
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
Pharmaceutical compositions and administration
Aqueous compositions of the present invention comprise an effective amount of
NSP4 (NSP4 114-135, NSP4 120-147, NSP4 112-175 or NSP4 112-150) protein,
polypeptide, peptide, expression vector, or cells containing the expression
vector, antibodies,
epitopic core region, inhibitor, and/or such like, dissolved and/or dispersed
in a
pharmaceutically acceptable earner and/or aqueous medium. Aqueous compositions
of gene
therapy vectors expressing any of the foregoing are also contemplated.
The phrases "pharmaceutically and/or pharmacologically acceptable" refer to
molecular entities and/or compositions that do not produce an adverse,
allergic and/or other
untoward reaction when administered to an animal as appropriate.
As used herein, "pharmaceutically acceptable carrier" includes any and/or all
solvents,
dispersion media, coatings, antibacterial and/or antifungal agents, isotonic
and/or absorption
delaying agents and/or the like. The use of such media and/or agents for
pharmaceutical
active substances is well known in the art. Except insofar as any conventional
media and/or
agent is incompatible with the active ingredient, its use in the therapeutic
compositions is
contemplated. Supplementary active ingredients can also be incorporated into
the
compositions. For administration, preparations should meet sterility,
pyrogenicity, general
safety and/or purity standards as required by FDA Office of Biologics
standards.
The biological material should be extensively dialyzed to remove undesired
small
molecular weight molecules and/or lyophilized for more ready formulation into
a desired
velucle, where appropriate. The active compounds may generally be formulated
for
parenteral administration, e.g., formulated for injection via the intravenous,
intramuscular,
sub-cutaneous, intralesional, and/or even intraperitoneal routes. The
preparation of an
aqueous compositions that contain an effective amount of the agent as an
active component
and/or ingredient will be known to those of skill in the art in light of the
present disclosure.
Typically, such compositions can be prepared as injectables, either as liquid
solutions and/or
suspensions; solid forms suitable for using to prepare solutions and/or
suspensions upon the
addition of a liquid prior to injection can also be prepared; and/or the
preparations can also be
emulsified.
The pharmaceutical forms suitable for injectable use include sterile aqueous
solutions
and/or dispersions; formulations including sesame oil, peanut oil and/or
aqueous propylene
43
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
glycol; and/or sterile powders for the extemporaneous preparation of sterile
injectable
solutions and/or dispersions. In all cases the form must be sterile and/or
must be fluid to the
extent that easy syringability exists. It must be stable under the conditions
of manufacture
and/or storage and/or must be preserved against the contaminating action of
microorganisms,
such as bacteria and/or fungi.
Solutions of the active compounds as free base and/or pharmacologically
acceptable
salts can be prepared in water suitably mixed with a surfactant, such as
hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid
polyethylene
glycols, and/or mixtures thereof and/or in oils. Under ordinary conditions of
storage a~id/or
use, these preparations contain a preservative to prevent the growth of
microorganisms.
The active compounds of the present invention can be formulated into a
composition
in a neutral and/or salt form. Phamnaceutically acceptable salts, include the
acid addition
salts (formed with the free amino groups of the protein) and/or which are
formed with
inorganic acids such as, for example, hydrochloric and/or phosphoric acids,
and/or such
organic acids as acetic, oxalic, tartaric, mandelic, and/or the like. Salts
formed with the free
carboxyl groups can also be derived from inorganic bases such as, for example,
sodium,
potassium, ammonium, calcium, and/or ferric hydroxides, and/or such organic
bases as
isopropylamine, trimethylamine, histidine, procaine and/or the like. In terms
of using peptide
therapeutics as active ingredients, the technology of U.S. Patents 4,608,251;
4,601,903;
4,599,231; 4,599,230; 4,596,792; and/or 4,578,770, each incorporated herein by
reference,
may be used.
The carrier can also be a solvent and/or dispersion medium containing, for
example,
water, ethanol, polyol (for example, glycerol, propylene glycol, and/or liquid
polyethylene
glycol, and/or the like), suitable mixtures thereof, and/or vegetable oils.
The proper fluidity
can be maintained, for example, by the use of a coating, such as lecithin, by
the maintenance
of the required particle size in the case of dispersion and/or by the use of
surfactants. The
prevention of the action of microorganisms can be brought about by various
antibacterial
and/or antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic
acid,
thimerosal, andlor the like. In many cases, it will be preferable to include
isotonic agents, for
example, sugars and/or sodium chloride. Prolonged absorption of the injectable
compositions
can be brought about by the use in the compositions of agents delaying
absorption, for
example, aluminum monostearate and/or gelatin.
44
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
Sterile injectable solutions are prepared by incorporating the active
compounds in the
required amount in the appropriate solvent with various of the other
ingredients enumerated
above, as required, followed by filtered sterilization. Generally, dispersions
are prepared by
incorporating the various sterilized active ingredients into a sterile vehicle
which contains the
basic dispersion medium and/or the required other ingredients from those
enumerated above.
In the case of sterile powders for the preparation of sterile injectable
solutions, the preferred
methods of preparation are vacuum-drying and/or freeze-drying techniques which
yield a
powder of the active ingredient plus any additional desired ingredient from a
previously
sterile-filtered solution thereof. The preparation of more, and/or highly,
concentrated
solutions for direct injection is also contemplated, where the use of DMSO as
solvent is
envisioned to result in extremely rapid penetration, delivering high
concentrations of the
active agents to a small tumor area. '
Upon formulation, solutions will be administered in a manner compatible with
the
dosage formulation and/or in such amount as is therapeutically effective. The
formulations
are easily administered in a variety of dosage forms, such as the type of
injectable solutions
described above, but drug release capsules and/or the life can also be
employed.
For parenteral administration in an aqueous solution, for example, the
solution should
be suitably buffered if necessary and/or the liquid diluent first rendered
isotonic with
suffcient saline and/or glucose. These particular aqueous solutions are
especially suitable for
intravenous, intrasnuscular, subcutaneous and/or intraperitoneal
administration. In this
connection, sterile aqueous media which can be employed will be known to those
of skill in
the art in light of the present disclosure (see for example, "Remington's
Pharmaceutical
Sciences" 15th Edition). Some variation in dosage will necessarily occur
depending on the
condition of the subject being treated. The person responsible for
administration will, in any
event, determine the appropriate dose for the individual subject.
The active peptides and/or agents may be formulated within a therapeutic
mixture to
comprise about 0.0001 to I.0 milligrams, andlor about 0.001 to 0.1 milligrams,
and/or about
0.1 to 1.0 and/or even about 10 milligrams per dose and/or so. Multiple doses
can also be
adrniiustered.
In addition to the compounds formulated for pareriteral administration, such
as
intravenous and/or intramuscular inj ection, other pharmaceutically acceptable
forms include,
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
e.g., tablets and/or other solids for oral administration; liposomal
formulations; time release
capsules; and/or any other form currently used, including cremes.
One skilled in the art recognizes that nasal solutions and/or sprays, aerosols
and/or
inhalants can be used in the present invention. Nasal solutions are usually
aqueous solutions
designed to be administered to the nasal passages in drops and/or sprays.
Nasal solutions are
prepared so that they are similar in many respects to nasal secretions, so
that normal ciliary
action is maintained. Thus, the aqueous nasal solutions usually are isotouc
and/or slightly
buffered to maintain a pH of 5.5 to 6.5. In addition, antimicrobial
preservatives, similar to
those used in ophthalmic preparations, and/or appropriate drug stabilizers, if
required, may be
included in the formulation. Various commercial nasal preparations are known
and/or
include, for example, antibiotics and/or antilustamines and/or are used for
asthma
prophylaxis.
Additional formulations which are suitable for other modes of administration
include
vaginal suppositories and/or pessaries. A rectal pessary and/or suppository
may also be used.
Suppositories are solid dosage forms of various weights and/or shapes, usually
medicated, for
insertion into the rectum, vagina and/or the urethra. After insertion,
suppositories soften,
melt and/or dissolve in the cavity fluids. In general, for suppositories,
traditional binders
and/or car-iers may include, for example, polyalkylene glycols and/or
triglycerides; such
suppositories may be formed from mixtures containing the active ingredient in
the range of
0.5% to 10%, preferably 1%-2%.
Oral formulations include such normally employed excipients as, for example,
pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium
saccharine,
cellulose, magnesium carbonate andlor the like. These compositions take the
form of
solutions, suspensions, tablets, pills, capsules, sustained release
formulations and/or powders.
In certain defined embodiments, oral pharmaceutical compositions will comprise
an inert
diluent and/or assimilable edible carrier, and/or they may be enclosed in hard
and/or soft shell
gelatin capsule, and/or they may be compressed into tablets, and/or they may
be incorporated
directly with the food of the diet. For oral therapeutic administration, the
active compounds
may be incorporated with excipients and/or used in the form of ingestible
tablets, buccal
tables, troches, capsules, elixirs, suspensions, syrups, wafers, axld/or the
like. Such
compositions and/or preparations should contain at least 0.1% of active
compound. The
percentage of the compositions and/or preparations may, of course, be varied
and/or may
46
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
conveniently be between about 2 to about 75% of the weight of the unit, and/or
preferably
between 25-60%. The amount of active compounds in such therapeutically useful
compositions is such that a suitable dosage will be obtained.
The tablets, troches, pills, capsules and/or the like may also contain the
following: a
binder, as gum tragacanth, acacia, cornstarch, and/or gelatin; excipients,
such as dicalcium
phosphate; a disintegrating agent, such as corn starch, potato starch, alginic
acid and/or the
like; a lubricant, such as magnesium stearate; and/or a sweetening agent, such
as sucrose,
lactose and/or saccharin may be added and/or a flavoring agent, such as
peppermint, oil of
wintergreen, and/or cherry flavoring. When the dosage unit form is a capsule,
it may contain,
in addition to materials of the above type, a liquid carrier. Various other
materials may be
present as coatings and/or to otherwise modify the physical form of the dosage
unit. For
instance, tablets, pills, and/or capsules may be coated with shellac, sugar
and/or both. A
syrup of elixir may contain the active compounds sucrose as ' a sweetening
agent methyl
and/or propylparabens as preservatives, a dye and/or flavoring, such as cherry
and/or orange
flavor.
EXAMPLES
The following examples are included to demonstrate preferred embodiments of
the
invention. It should be appreciated by those of skill in the art that the
techniques disclosed in
the examples which follow represent techniques discovered by the inventor to
function well
in the practice of the invention, and thus can be considered to constitute
preferred modes for
its practice. However, those of shill in the art should, in light of the
present disclosure,
appreciate that many changes can be made in the specific embodiments which are
disclosed
and still obtain a like or similar result without departing from the spirit
and scope of the
invention.
Example 1
NSP4
NSP4 was purified from recombinant-baculovirus pAC461-G10 infected Spodoptera
frugiperda (Sf~3) cells expressing gene 10 by FPLC on a QMA anion exchange
column as
previously described (Tiara et czl., 1994 and Tiara et al., 1995), and with an
additional affinity
purification step on a column containing anti-NSP4 antibodies. Different NSP4
preparations
of >70% and 90% purity gave the same biologic results. The protein was sterile
based on
47
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
bacteriologic culturing in L-broth incubated at 37 C for one week, and lacked
endotoxin
based on testing by the limulus amebocyte lysate (LAL) assay (Levin 1968 and
Novitsky
1984). VP6 was purified to >95% purity from recombinant-baculovirus
pAc461/SAll-G6
infected S~ cells by gradient centrifugation as previously described (Zeng et
al., 1996).
Both proteins were lyophilized and diluted in sterile PBS to a final volume of
50 ~,l per dose,
regardless of the route of administration. .
Example 2
Synthetic peptides
Synthetic NSP4-specific and control peptides utilized in this study were
originally
selected based on algorithms which predict surface potential (Parker et al.,
1986), turn
potential (Pt) (Chou 1978), and amphipathic structure (Margolit et al., 1987).
A block length
of 11 was used and an amphipathic score (AS) of 4 was considered significant.
Sequences
were selected based on the high predicted propensities for folding into
amphipathic helices
and reverse turns, because small peptides which typically laclc any folding
pattern in an
aqueous environment can fold into an ordered secondary structure resembling
the nascent
protein if the structural propensity is high (Dyson et al., 1988; Dyson et
al., 1991; Dyson et
al., 1988; Dyson et al., 1995; Yao et al., 1994; Waltho et al., 1993; Dyson et
al., 1992;
Wright et al., 1988).
Peptide sequences used in this study include: NSP4 114-135 (Both et al.,
1983),
(DKLTTREIEQVELLKRIYDKLT, SEQ.ID.NO:1), AS=35; a peptide from the amino-
terminus of NSP4, NSP4 2-22 (EKLTDLNYTLSVITLMNNTLH, SEQ.ID.NO:2), AS=14;
an extended highly amphipathic peptide, NSP4 90-123
(TKDEIEKQMDRVVKEMRRQLEMIDKLTTREIEQ, SEQ.ID.N0:3) AS=71; a mutated
NSP4 114-135 peptide, mNSP4 131K (DKLTTREIEQVELLKRIKD KLT, SEQ.ID.N0:4)
AS=31; and a peptide from the COOH- terminus of the Norwallc virus capsid
protein having
a centrally located tyrosine residue (Jiang et al., 1990), NV 464-483
(DTGRNLGEFKAYPDGFLTCV, SEQ.ID.NO:S) AS=41 (Table 1), and NSP4 120-147
(EIEQVELLKRIYDKLTVQTTGEIDMTKE, SEQ.ID.N0:6) AS=35.0, NSP4 112-175
(MIDKLTTREI EQVELL
I~RIYDKLTVQTTGEIDMTKEINQKNVRTLEEWESGKNPYEPKEVTAAM,
SEQ.ID.N0:9) and NSP4 112-150 (MIDKLTTREIEQVELLI~RIYDK LTVQTTGEm
MTKEINQ, SEQ.ID.N0:10).
48
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
d' N 01 d'
dM' O ~ N
N N ~' N N
w ~ ~ ~ o
b
'-'
b
~ a O
C3
:
N N ~ 00 00
M r-1 r-I r-1 O V7 ~U
7
'
~ .
q
M +~
O .~.',
w
0
~b
. U C/~
'~
N O1 ~O ~Y
M O ~ ~ ~ ~ 4-~
M d' ,~
.
~
m ~ O
v~
r1 ~ 'C
+~O
O
C~ 'n ~ O
b bD
CC~ ~ . w
~ F~
E'~ O a~
O O
~ ~
~
O ..r
b
, cct
O
H z
~' w ~ ~
~ ~, a ~
a a NO ,
'
w , G
N a~
N v~
O ~
~ U
a~
as
H rx w
~ o
w
~
H ~ H
A ~ ~
v~ ~ ~
U U c~
',~ .'~
H c
~
U
~ b N
p
.IjN S~
M N
N ~ M o0 ~
b
N O '-' ~ P v~
~ .~ I
v~
s~
N 01 ~ Z t~.
~ ~
~n P..~
z z z z
N cn
~I-
49
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
All peptides were synthesized by the University of Pittsburgh Peptide Core
Facility
employing Fmoc chemical strategy and standard protocols (Carpino 1970).
Coupling and
deblocking efficiencies were monitored by the ninhydrin colorometric reaction
(Kaiser, et al.,
1970). Peptides were cleaved from their solid resin support and separated from
organic
contaminants by multiple cold ether extractions, and conventional gel
filtration
chromatography (Sephadex G-25). The final peptide product was characterized by
reverse-
phase HPLC (Deltapak C4, Waters) and plasma desorption mass spectroscopy
(Johnson et
al., 1986). Only those peptides with the correct theoretical mass and 90% or
greater full-
length product were employed in these studies. Prior to use, peptides were
further purified
either by HPLC on a semi-preparative, reverse-phase C18 colurmz (uBondapak,
Waters) or by
multiple elutions from a conventional gel filtration column (1.5 mm X 40 mm).
Peptide
purity was conf rmed prior to inoculations by gel filtration chromatography
(Protein-Pak 60
column, 10~.m, Waters) on a Waters HPLC unit. The elution profiles were
monitored by UV
absorption (Lambda-Max LC-spectrophotometer, Waters) at 220nm and recorded by
a 745
Data Module (Waters). The elution buffer was PBS, pH 7.2, and the flow rate
0.5 ml/min.
Sterility was confirmed as described for NSP4 protein.
Example 3
Glutaraldehyde cross-linking of synthetic peptides
Peptides were cross-linked to themselves or to the carrier protein, keyhole
limpet
hemocyanin (KLH), by glutaraldehyde in a single-step coupling protocol
(Reichlin 1980).
Briefly, the peptide immunogen was coupled to KLH at a ratio of 100 nmol
peptide: 1 nmol
KLH or to itself at a 1:1 ratio by the addition of glutaraldehyde to a final
concentration of
0.4%. The reaction was quenched by the addition of 1M glycine (Cf--20mM). The
cross-
linked peptides were extensively dialyzed against sterile PBS prior to use.
Example 4
Antibody production
NSP4 114-135 peptide-specific antiserum was generated in CD1 mice and New
Zealand white rabbits by immunization with peptide cross-linked via
glutaraldehyde to the
protein carrier KLH, as described above. The first inoculum was emulsified in
Freund's
complete adjuvant, whereas all subsequent inoculations were prepared in
incomplete
Freund's adjuvant. Rabbits were injected intramuscularly (1M, once in each
hip) and
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
subcutaneously (SC) across the back of the neck. Boosting doses of emulsified
antigen (100
mnol of peptide) were done every 4 wk for a total of 5 immunizations. Mice
were
immunized every three weeks by the IM, SC and IP routes. Preimmunization and
postimmunization sera were evaluated by peptide ELISAs (titer of 400-3200) as
previously
described (Ball et al., 1994) and by Western blot analyses.
Example 5
IP and IL administration of protein and peptides
Purified NSP4 protein, peptide alone, or cross-linked to itself, were
administered to
young (6-10 days) and older (11-25 days) outbred CDl or inbred Balb/C mice,
and outbred
Sprague-Dawley rats by the intraperitoneal (IP), intraileal (IL),
intramuscular (IM),
subcutaneous and oral routes. The peptide or protein inocula were diluted in
sterile PBS to a
final volume of 50 ~.1 per dose, regardless of the route of administration or
inoculum. A 30 G
needle was employed for the IP and IL delivery of the inocula. Peptide was
delivered orally
to young mice by gavage using a PE-10 polyethylene flexible tubing
(Intramedic, Becton
Dickinson) and food coloring. For the surgical introduction of the peptide or
protein via the
IL route, animals were anesthetized with isofurane (Anaquest), a small
incision was made
below the stomach, the inocula were directly injected into the upper ileum,
and the incision
was sealed with polypropylene sutures (PROLENE 6-0, Ethicon). The pups were
isolated,
kept warm, and closely monitored for a minimum of 2 hrs prior to returning
them to their
cage.
Example 6
Monitoring of diarrhea induction
Diarrhea induction by the NSP4 protein and peptides was carefully monitored
for 24
hrs following the inoculations. Each pup was examined every 1-2 hr for the
first 8 hr and at
24 hr post inoculation by gently pressing on the abdomen. Diarrhea was noted
and scored
from 1 to 4 with a score of 1 reflecting unusually soft, loose, yellow stool,
and a score of 4
being completely liquid stool. A score of 2 (mucous with liquid stool, some
loose but solid
stool) and above was considered diarrhea. A score of 1 was noted, but was not
considered as
diarrhea. The scoring was done by a single person and the pups were coded
during analysis
of diarrhea. Other symptoms monitored included lethargy, coldness to the
touch, and ruffled
coats in older animals.
51
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
Example 7
Analysis of chloride secretion responsiveness to NSP4114-135
in the intestinal mucosa of mice
Unstripped intestinal mucosal sheets from 19-22 and 35 day old mice were
analyzed
for chloride secretory responsiveness to NSP4 114-135. Short-circuit currents
(Isc) were
measured across unstripped intestinal mucosal sheets from 19-22 and 35 day old
CD1 mice
using an automatic voltage clamp (Bioengineering, Univ. of Iowa) as described
previously
(Sears et al., 1995 and Morris et al., 1994). The mid-ileum of the mouse
intestines was
utilized. The unstripped mucosal sheets taken from the intestine were placed
into modified
Ussing chambers with 0.12 cm2 apertures (machine shop, UTHSC) and
transepithelial
potential (Vt) was registered by 3 M KCl agar bridges connected to balanced
calomel half
cells. The transepithelial current required to clamp Vt to 0 was passed
through Ag-AgCl
electrodes connected to the 3 M KCl bridges. All experiments were performed at
37 C in
bicarbonate Ringers solution gassed with 95% O2-5% COZ by airlift circulators
as previously
described (same as above). The mucosal bath contained sodium-free (N-methyl-D-
glutamine) substituted Ringers to minimize the effects on Isc of cAMP
stimulated
electrogenic Na glucose co-transport across the small bowel (Grubb 1995).
Following
temperature and ionic equilibration, basal Isc measurements were taken and
intestinal
mucosal sheets were challenged with cross-linced peptide (either NSP4 114-135,
NSP4 2-22,
or mNSP4 I3IK), the calcium-elevating agonist carbachol (Cch), or the cAMP-
agonist
forskolin (FSK). Bumetamide sensitivity was tested and confirmed the chloride
secretory
response.
Example 8
NSP4 protein induces age-dependent diarrhea in mice
Whether administration was IP or intraileal (IL), diarrhea was observed within
1 to 4
hr post inoculation, typically continued for up to 8 hr, but occasionally
persisted for 24 hr.
Purified NSP4 (0.1-Snmol) was administered by the IP route to 6-7 and 8-9 day
old CD1
pups. In 6-7 day old CD 1 pups, IP administration of 0.1 nmol of NSP4 induced
diarrhea in
60% of the mice, whereas no disease was induced in 8-9 day old mice with the
same
concentration of protein (FIG. 1). IP administration of 1 nmol of NSP4
resulted in 100% of
the 6-7 day pups with diarrhea, and 60% of the 8-9 day old mice with disease.
A larger dose
of 5 nmol of NSP4 induced diarrhea in 90% of the older (8-9 day) mice.
Additional clinical
52
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
symptoms included lethargy and coldness to the touch, which were observed in
the majority
of treated aiumals with diarrhea of all ages. The induction of diarrhea by
NSP4 was shown to
be specific for this protein as administration of the same volume of buffer or
VP6 had no
effect.
IL administration of 0.5 mnol of purified NSP4 protein resulted in disease in
100% of
the CD 1 pups (8-9 day old mice) within the first 2 hr post inoculation,
whereas no diarrhea
was observed in 17-18 day old pups (Table 2, FIG. 1).
53
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
N O M N O O O
."
A
O
."
O O
O
O O ~ ~ V07 ~ N O O
O
U
~/j tP7 tn ~ V7 tn~(i tn tn
b Y d W' ' f d' d' ' d.
l'
~ r w "
V'
y ~ d. d. d.d. '~' d, d.
~ ~ ~ f~ ~ %
z o z z C z C z z z z z
I~ I~
z z
~
_
M
0
n1 vn n. y.y ..irr n.1 i..in.l
l~ 01 r1 e1 r1 ~ ~ U'701
~
~D 00 e-IU1 r-I l~ N N 00
v G G y y y y ~ y
..,~, ."
..fir..~.i
..~.i
A U U V
,~ ,~ ,-,
,a ~,a .r~
A A A A ,
A
~.
54
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
Thus, the. response to NSP4 was age- and dose-dependent in CD1 pups. In
addition,
the induction of diarrhea by NSP4 was specific, as administration o~ the same
concentration
of purified rotavirus VP6 or the same volume of buffer had no effect (FIG. 1).
The effect of
IP and IL delivery of NSP4 protein in mice is the same. Intramuscular (IM)
inoculation of 1
nnlol of purified NSP4 produced no ill effects. Subcutaneous and oral
administration of NSP4
also produced no ill effects.
Additional data showing a dose response in 6-7 day old CD 1 pups is presented
in
FIG. 2. The amomlt of peptide administered is shown in nanomoles and
micrograms. 0.04-
1.0 mnols (1-25 pg) of purified NSP4 was administered to 6-7 day old CD-1 pups
by the IP
route. A correlation between increasing incidence of diarrhea and increasing
dose was seen
(FIG. 2) over the range tested. The highest tested dose (1.0 nmol=25 p.g)
induced diarrhea in
all mice tested (10 of 10).
Example 9
NSP4114-135 peptide induces diarrhea in mice
The NSP4 114-135 peptide has an AS of 35, is localized in the cytoplasmic
domain of
NSP4, and mobilizes intracellular calcium in eul~aryotic cells (Tian et al.,
1994 and Tian et
al., 1995).
Following IP administration of 0.1 to 50 nmol of the NSP4 114-135 peptide, a
similar
disease response was noted in 6-7 day old CD1 outbred pups with 30-40%
diarrhea induction
(FIG. 3). The percentage of CD1 pups with diarrhea increased to 60-70%
following the IP
delivery of 100-400 nmol of NSP4 114-135 and 89% of pups had diarrhea
following
administration of a dose of 500 nmol of peptide. Induction of disease in 100%
of the CD1
pups was not aclueved; doses exceeding 500 mnol were not administered since
the volume of
each dose was limited to 50,1. These data indicate the disease response in CD1
mice can be
divided into three groups based on the dose of the NSP4 114-135 peptide, 1)
less than and
equal to 50 nmol (1mM) resulting in 30-40% of the animals with disease, 2) 100-
400 nmol
(2-8mM) yielding disease in 60-70% of the animals, and 3) 500 nmol (lOmM)
above
inducing diarrhea in at least 89% of the young mice.
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
Diarrhea was induced in 100% of the 6-7 day old Balb/C pups with lower
concentrations (only 50 mnol) of peptide (FIG. 4), and diarrhea was observed
in 80% of the
Balb/C mice given 0.1 nmol (2~.M) of NSP4 114-135. Hence the Balb/C pups
appeared more
sensitive to the effects of NSP4 114-135.
Doses exceeding 50 nmol (1mM) of NSP4 114-135 peptide were sufficient to
induce
diarrhea in the majority of young mice when administered by the IP route. The
diarrhea was
observed within 1 to 4 hr post inoculation and typically continued for up to 8
hr, but
occasionally was present for 24 hr. The severity of diarrhea typically
increased with time.
That is, a mouse with a diarrhea score of 1 in the first hr post inoculation
would have a
diarrhea score of 4 in the next hr. Various degrees of lethargy were noted
following the
administration of peptide and this was most pronounced at 3 to 4 hr post
inoculation. The
lethargy was accompanied by the pups being cold to the touch and was age-
dependent. The
severity of the induced diarrhea was greater in the Balb/C pups. No symptoms
were noted
with control peptides (NSP4 2-22, NV C-terminus) or PBS administered to the
same age and
species of mice.
Example 10
NSP4 120-147 peptide induces diarrhea in mice
A peptide corresponding to amino acid residues 120-147 of NSP4 was prepared
and
tested in 5-7 day old pups. When a dose of 100 nmols was administered, all (5
of 5) animals
exhibited severe diarrhea. A dose of 5 nmols induced diarrhea in 7 out of 8
animals (88%).
This demonstrates that other peptides derived from NSP4 can be prepared and
screened to
find the peptide with the highest activity. It is well within the ability of
one of ordinary skill
in the art to synthesize and screen a library of overlapping peptides that
represents the entire
sequence of the NSP4 protein in order to locate peptides with biological
activity. One skilled
in the art can readily appreciate that both the length of the peptides, and
the number of
residues that overlap in adjacent peptides, can be varied at the discretion of
the practitioner
without deviating from the spirit of the present invention.
Example 11
Diarrhea induction in CDl and Ealb/C mice by cross-linked NSP4114-135
The NSP4 114-135 peptide was cross-linked to itself by glutaraldehyde and
administered to young mouse pups by the IP route to determine if the diarrhea
induction was
56
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
affected by structure or oligomerization. Diarrhea was induced in the majority
of the CD1
pups at a lower dose of NSP4 114-135 when the peptide was cross-linked to
itself when
compared to the peptide alone (FIG. 7A and FIG. 7B). One nmol of cross-linked
peptide
induced diarrhea in 80% of the CD1 pups which increased to 90% with 250 nmol
of cross-
linked NSP4 114-135. As illustrated in FIG. 5 and FIG. 6, doses at or above 1
nmol (20~M)
of cross-linked peptide were sufficient to elicit a response in the majority
of the CD1 pups.
Increasing the dose above 1 nmol of cross-linked NSP4 114-135 had little
effect, indicating
the diarrheal response could not be increased with increased amounts of
synthetic peptide, or
that the response, once stimulated, could be saturated or additional
stimulation had no effect.
Similar to the response in CD1 mice, diarrhea induction in 100% of the Balb/C
pups
was achieved with a lower dose (10 nmol, 200~,M) of cross-linked peptide when
compared to
the peptide alone (FTG. 7A and FIG. 7B). In addition, the lethargy and
coldness to the touch
were more severe and lasted longer in animals that received the cross-linked
peptide. Cross
linked NSP4 2-22 and NV C-terminus peptides were administered as controls and
did not
induce symptoms in young mice.
Induction of disease at a lower dose and with greater severity with the IP
administration of cross-linked NSP4 114-135 suggests that cross-linking either
stabilizes the
peptide, oligomerizes the peptide, or results in a conformation more closely
resembling the
native protein. These data suggest structure may be important for disease
induction.
Example 12
Cross-linked NSP4 114-135 peptide also induces diarrhea in young rats
The NSP4 114-135 peptide was tested in a second species, the Sprague-Dawley
rat to
determine whether the disease response induced by this peptide was only
effective in young
mice. IP inoculation of 100-250 nmol of cross-linlced peptide induced diarrhea
in 78% of
young (6 days) rat pups and in none of the older (10 day) rat pups (FIG. 5).
No disease was
observed in the same age rodents administered control peptides. The response
in rats was
slower than that observed in mice, taping from 6 to 12 hr before the onset of
diarrhea was
noted, compared to 2 to 4 hours post inoculation for the mice, and required a
higher
concentration of peptide to observe disease. However, the induced diarrhea and
lethargy in
the young rats frequently persisted for up to 48 hr. These differences may
reflect the
57
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
difference in size and intestinal transit time between the rat and mouse or
species (genetic)
variation.
Example 13
IL delivery of NSP4 peptide
IL administration of I20-240 nmol of cross-linked NSP4 I14-135 induced
diarrhea in
90% of young (6-7 days) rat pups. Analogous to the response of young rats
following the IP
administration of cross-linked peptide, the onset of diarrhea was slower than
that seen in the
mice, taking from 6 to 12 hr, but lasted for a greater length of time (up to
48 hr). The surgical
introduction of 10 nmol (200~.M) of cross-linked peptide induced diarrhea in
100% of the
young (8-9 days) Balb/C pups, identical to the induction of diarrhea following
IP delivery
(Table 2). The age-dependence of the diarrhea response noted with the IP
administration of
cross-linked NSP4 114-135 was maintained with the IL administration of cross-
linked
peptide. Only one-third of the 11-12 day old Balb/C mice had diarrhea when
administered 10
nmol of cross-linked peptide by the IL route, and none of the 15-17 day
animals had diarrhea.
In addition, older CDl mice (11-12 and 25 days) had no ill effects from the IL
delivery of 50-
200 nmol (1-4 mM) of cross-linked peptide (FIG. 6, Table 2). An equal
concentration of
cross-linked NSP4 2-22 peptide or an equal volume of PBS, when surgically
introduced in
both young and older rodents, had no ill effects (data not shown).
Hence, the effect of IP and IL delivery of NSP4 peptide in rodents was
equivalent.
Example 14
Diarrhea induction is age dependent
Between 100 and 300 nmol of NSP4 114-135 peptide, alone or cross-linked, was
administered by the IP route to different age outbred mice and rats. Diarrhea
was observed in
the young mice within 2 to 4 hr post inoculation, whereas reduced or no
symptoms were seen
in older (11-12 or 15-17 days) animals (FIG. 5 and FIG. 6). With IP
administration of
peptide alone, disease was induced in 60% of the 6-7 day old CDl pups with no
symptoms
noted in the 11-12 and 15-17 day old mice. IP administration of cross-linked
peptide resulted
in 90% diarrhea induction in 6-7 day old CD1 pups, 30% disease in 11-12 day
old pups, and
only 10% disease in 15-17 day old mice.
58
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
A comparable age dependence was observed with the Sprague-Dawley rats when
cross-linked peptide was administered by the IP route. Diarrhea was detected 6
to 12 hr post
inoculation in 78% of the young (5-6 day) rats while no disease was seen in
the 10 day old
rats given a similar dose of cross-linked peptide (FIG. 5 and FIG. 6). Thus an
age
dependence, similar to what is seen in a natural infection, is seen with the
NSP4 114-135
peptide.
Example 15
Induction of diarrhea is dose dependent
Doses of 0.1-500 nmol of a peptide were administered IP to 84 CD1 pups (6-7
days
old; FIG. 3 and FIG. 4). The disease response to the NSP4 114-135 peptide was
dose-
dependent (x2t,.e"a=9.98, p=0.0016) with a DD50 (50% diarrheal dose) of 79
ntnol (Collins et
al., 1988).
Example 16
Specificity of the diarrhea response to peptide NSP4 114-135
Specificity of the diarrhea induction by the NSP4 114-135 peptide was
confirmed by
the administration of a panel of control peptides to young mouse pups (Table 1
and Table 3).
Mutant peptide mNSP4 131K, in wluch the tyrosine at position 131 of NSP4 114-
135
is replace with a lysine did not induce diarrhea (0/11), indicating the
importance of this
tyrosine residue in the induction of diarrhea. Neither did NSP4 2-22 or NV 464-
483 cause
diarrhea, 0111 and 0/10, respectively. NSP4 90-123, which overlaps the 114-135
peptide by 9
residues, induced diarrhea in only 20% (2/10) of the mice tested (Table 3).
The percentage of
diarrhea induction increased to 50% when the NSP4 90-123 peptide was
crosslin~ed. Cross-
linked mutant (m)NSP4 131k peptide induced diarrhea in 2 of 10 mice, while
cross-linked
NV 464-483 did not cause disease. Thus, the response to peptide alone appears
to be directed
to a region of NSP4 inclusive of residues 114-135.
59
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
a~
O N O N .--~ O ~ l~ O ~ O
O N O O N ~ O O
b
O O
..,
a~
..~dG
O ~
~" i"~ o
~ .b ~s
o P~
ri ~
~ o ~° o o N ° 0 0 0
E-~ ~ ~ o ~ G
o A o ø'
..''~~' .p .N b
0
~b
c~
NNp~p~b
M r'' d' ~
i d' r1 N ~ i ~
V7 ,.~~ r, ,9 ~' d' M Wp
b d' p~.~ ~ M ~ N ~ ~ ~ 'd ~ Z o
_,~ r, N Z~ O ~ cn
N z b Pa ~ P.~ °~ d' 'b
z
z ~ ~ ~ ~ . z ~ ~ b
0
~n ~ ~ ~ o
U U U
3
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
Example 17
Administration of peptide NSP4114-135 results in stunted growth
Animals given peptide three times per day for two days showed a rapid onset of
severe diarrhea followed by stunted growth. The weight of these animals was 20-
30% lower
for three weeks after administration of peptide (FIG. 9). These results mimic
characteristics
of rotavirus disease in animals and children, including the fact that both may
show decreased
growth rates after multiple infections.
Example 18
Antiserum to NSP4114-135 peptide blocks induction of diarrhea
In the absence of antibody, IP delivery of 50-100 nrilol of NSP4 114-135
peptide
induced diarrhea in 67% of the mice. IP inoculation of NSP4 114-135 peptide-
specific
antiserum 5 mins prior to IP delivery of peptide (50-100 nmol) resulted in a
90% reduction of
disease. IP administration of normal rabbit serum prior to peptide did not
block the diarrhea.
Example 19
NSP4 antibodies protect against virus-induced disease
The potential of NSP4 antibodies to protect against virus-induced disease was
tested
by challenging pups, born to dams which were immunized with the NSP4 114-135
peptide or
a control peptide, with a high dose of infectious SA11 virus, FIG. 8, left
hand side. Diarrheal
disease in pups born to dams immunized with the NSP4 114-135 peptide was
significantly
(Fisher's exact test) reduced in severity, duration, and in the number of pups
with diarrhea
(Table 4). The NSP4 2-22 peptide was used as a control peptide, as it does not
induce
diarrhea in pups.
61
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
aio
0
a~
0
~cd 'n o .
G
.~~',M N .
U
,
o
p.,
O yn
. M
V b
;'
V: d.
O ~ '~
L/~
z
~, a~ ~~
N
~
M
U
~
.
3
v ~ b
i
.
et ~ .~ " p.
o
o a
,~
o
0
b ~ ~~ ~ N O
,~ .s-~''' o o O o
~' \ \ s3,
cd v ,-~
., M l~
. ~ w Q
~O ~
G ii ~ ~ i M
, .
~ N V
a~
M ~
~ r
.w '~
c~
."
a~
a>
o
O ~
O
~ by
~
~ o a ~
~ O
~ O N f
r., ~
.-i dW ~F ~N
a ..r uU7
~
H~
O
~
S~,
r, DCp
a>
i~ N d' v~
N ;
N ~ N~
ci~
P
z z
62
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
In another experiment, young mouse pups were infected with SAl1 virus, and
NSP4
antiserum or control antiserum was orally administered every 4-6 hours for 60
hr, FIG. 8,
right hand side. The pups administered NSP4-specific antibody had
significantly reduced
diarrheal disease compared to animals given no treatment, rabbit pre-immune
serum or
normal rabbit serum (NRS), (Table 5). These data show the potential of NSP4
antibodies to
bloclc rotavirus-induced disease.
63
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
0 0
\ \
o,0 0 0 ~ ~ o
.~ ~ ~ 0 0 0 0 0
~o
O M O
~
~
H N M O 0 0 0 O
I
0 0 ~ 0 0 0 0
\ \
a ~ ~ O
wr
+,
.Cs
a
M
C
~ ~
l r
i
~
.~ H f h f N O O
ed r
~
A V ~ ~ ~ '"' 01
~ ~ \
y ~ l ~ M M .-.-
0 a
W ~
y 00M 00~ 0000 M
'~
F.~
~
~ l~N l~N l~t~ N
' N N N N N N N
O
~
~ O
.,.,
0 0 0 0
~ r~ n
O O O O o 0 0
O O O O O ~n N
'~ v
c~i
L, ,
~ es ~ ~
~
cH y O M ~3E
. ., 00 .-1~ Q1
H i ~ o ~ ~ N N
P~
.-aN -~N ~ N ~ N
4
w
a~ Q,
cn
z
H
64
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
Example 20
Electrophysiological analyses
The effects of a peptide, and known Ca2+ - and cAMP-elevating agonists were
tested
on unstripped mouse intestinal mucosal sheets in modified Ussing chambers
(Giannella et al.,
1983; Curne et al., 1992; Field et al., 1978 and Forte et al., 1992). Addition
of forskolin
(FSK, CAMP agonist) and carbachol (Cch, cholinergic agonist which mobilizes
Ca2+) to
normal mouse deal mucosa resulted in measurable elevations in Cl- secretory
short circuit
current (Isc, Table 6). Addition of either 5 ~,M of NSP4 114-135 peptide
(cross-linked to
itself for enhanced stability) or 5 ~,M of Cch to mucosal sheets of 19-22 day
old CD1 mice
induced small (3 or 9 ~.A/cm2, respectively) and transient (1-2 min) increases
in Isc. When
the mucosal sheets were exposed to 5 ~,M of the cAMP-mobilizing agoiust, FSK,
larger
increases in Isc (44 ~,A/cm2 ) were elicited that reached sustained levels
within 2-3 min.
After FSK pretreatment, challenge of the mucosa with either peptide or Cch
resulted in much
larger increases in mucosal Isc (64 or 63 ~,A/cm2, respectively); both the
peptide and Cch
potentiated the response to FSK. All of the responses to agonists were
sensitive to
bumetamide and treatment of deal mucosal sheets with cross-linked control NSP4
2-22
peptide did not induce a response. Addition of Cch to 19-22 day old mouse
mucosal sheets
which had been pretreated with peptide alone, or peptide in combination with
FSK, had
minimal or no additional effect on Isc. This subsequent loss of sensitivity to
the Ca2+ -
elevating agonist (Cch) after peptide pretreatment suggests that the NSP4
peptide increases
Isc through changes in intracellular Ca2+ ([Ca2+];). Addition of Cch to mucosa
from a 35 day
old mouse again elicited a small (14 ~,A/cm2) and transient (1-2 min) response
that
potentiated the effect of FSK (64 ~Alcm2), whereas there was no or minimal
increase in Isc
when the NSP4 114-135 peptide was added alone or with FSK to the 35 day old
mouse
mucosal sheets (Table 6).
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
U
.
b
O
,d O
bA
N cd
N 4-,
O
_ b_
V7 V7 'd'
ISI I~ '~ b
~r a a a ~O
N
0
M I _-~
w a
v d' ~ O ~ d' °'
-li
O~O 0~0 ~' ~! ~ ~: 'd
V ~ ~ I-~-~ I~' I~'' I~''
H ~ a ~r a 'r a ~ N
r1
A ~ t~ N O
eU ~i I O N O
O ~ ~, .~ +I M d~" ~ O can
y d' 41 M lp ~O ~~ ~ +~.. ~ N
H C~
a>
O
O ~ .~ O O
V ~ .~'' ~ ~ yak,
bD ~ y.
b~ ~_~
es ø, ~ ''°'r .~J °' II
N ~ N . ~ y ,+'~- t~
y ~ O ~~ ~ y U O
v In ~ .~ ~ O 4-i
.N M ~" 'b m 4~
O ,_, .-~ w 'd ai
m-. o ~ b a> b
U i~..i ~ ~ _N ~ '~ b
H O'. ,., "~ b bD ~~' ~ u~'
N N S~
r"' b
Vi ~ ~' ~ ~ d' ~ M ~ U .-O ..~..
c m ~ ~ v ~ ~°,~.9 ~ ~ 3
U z
I I -E' ~ ~ ~ ;?', ~ p.
'U'~ d. ~ ~ N ~ '3
O
a
,.fl Pi ~ ~ ..~ ~ ~ ,~ a, 3
w ~ Z w w
p oo v ~ dW
M
~~~,~~M
~ ~.,, 1 ~ ~ ~
~~; ~ ~~ ~,
H,j~.oZw°
cad ~ .-fl t~ U b
66
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
The electrophysiological responses from 19 day old mice initially seem
paradoxical to
the biological data since measurable secretion was .not observed as diarrhea
in this age
animal. Diarrhea likely was not seen in these older animals because of fluid
reabsorption by
the colon. This hypothesis was tested by IL administration of 200 nmol of NSP4
114-135 or
control peptide to 19 day old pups. At 4 hrs post inoculation, the mice were
sacrificed and
the intestines were tied off, removed, weighed, and the length measured. The
pups given
NSP4 114-135 peptide showed significant fluid accumulation when compared to
the control
pups although no diarrhea was seen in any animals.
It is anticipated that younger mice would show a greater increase in Isc than
that seen
in the 19 day old mucosa. However, intestinal mucosa from younger mice (<19
days) could
not be mounted efficiently into the Ussing chambers due to their small size;
such experiments
in very young mice will require the development of new methods to measure Cl-
secretion iya
vitf°o. Nonetheless, the NSP4 114-135 peptide did not augment secretion
in 35 day old mice,
correlating the age-dependence seen ih vivo.
Example 21
Live rotavirus and NSP4 cause diarrhea in CFTR knock-out mice
Cystic Fibrosis is caused by a defect in the gene that codes for the cAMP-
activated
chloride chaimel called CFTR. As a result of the defect, the CFTR channel is
defective and
chloride secretion -- and hence water secretion -- is greatly diminished.
Without sufficient
secretion of water, membranes accumulate excessive amounts of mucous and
eventually
become obstructed.
Peptide or virus was admiustered to 5-7 day old CFTR knock-out mice -- mice
homozygous for a mutation that disables the CFTR coding region -- and got
diarrhea in 100%
of the cases for virus and cross-linked peptide or in 80% of the animals given
100 rimoles of
non-crosslinked NSP4 114-135.peptide. This demonstrates that NSP4 stimulation
of chloride
secretion through a Caz+-dependent channel can compensate for the lack of
secretion through
the defective cAMP-dependent CFTR channel.
67
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
Example 22
HIV gp120 causes diarrhea in mice
Human immunodeficiency virus (HIV) is associated with wasting or Slim disease.
To
determine whether the HIV glycoprotein 120 (gp120) is an enterotoxin, 6-7 day
old Balb/C
mouse pups were inoculated with purified gp120. Diarrhea was observed in 100%
of the
animals. Other proteins of HIV or other retrovirus or other proteins of other
viruses may be
found to have similar functional activity - i. e., to directly induce
diarrhea.
Example 23
Identification of small molecule inhibitors of NSP4/receptor interaction.
The above data demonstrate that effective treatment of rotavirus-induced
diarrhea can
be accomplished through inhibition of NSP4's interaction with its receptor.
Identification of
small molecule inhibitors of NSP4 is well within the ability of the ordinary
practitioner
according to known techniques. Small molecule inhibitors are known in the art
to refer to
any ligand which can bind to a target molecule with sufficient affinity to
inlubit the target
molecule's activity. Libraries of small molecules, such as random peptide
libraries, random
oligonucleotide libraries, and pharmaceutical drug libraries, are available
either according to
known techniques or cormnercially, and may be quickly and easily screened
against a
purified target molecule for small molecules that bind with high affiuty to a
target molecule.
Examples include the "FliTrx Peptide Library," (Invitrogen) and the SELEX
technology.
Example 24
Construction of attenuated rotavirus strains by incorporation of a selected
NSP4 amino
acid sequence
The sequence of gene 10, the gene encoding NSP4, was determined for a pair of
virulent and tissue culture attenuated porcine rotavirus strains. Double
stranded RNAs were
extracted from an intestinal homogenate from a piglet infected with a virulent
strain of
porcine rotavirus (OSU-v, SEQ.ID.N0:7) and from a piglet infected with a
tissue culture
attenuated OSU virus (OSU-a, SEQ.ID.N0:8). Gene 10 from the dsRNAs was
amplified by
RT/PCR using primers from the SA11 gene 10 sequence. cDNAs from these two
strains
were cloned and sequenced. Comparisons of the gene 10 sequences of these two
strains and
other rotavirus strains have suggested that the amino acid sequence between
amino acids 131
to 140 are important in pathogenesis. The amino acid sequence of the NSP4
protein from the
68
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
attenuated strain (OSU-a) was compared to that of the virulent strain (OSU-v)
and the results
are presented in FIG. 10. The positions at which the two sequences differ are
shown in bold.
Mice infected with virulent virus develop diarrhea while those infected with
attenuated virus
do not.
Gene 10 encoding NSP4 protein from each of these two strains has been cloned
and
expressed in a baculovirus expression system and purified. The purified NSP4
proteins were
tested for their ability to induce diarrhea in mouse pups. The NSP4 protein
from the virulent
strain causes increased intracellular calcium concentration and induced
diarrhea while that of
the attenuated strain did not. These results indicate that avirulence is
associated with
mutations in gene 10 and indicate that certain amino acid positions of the
NSP4 protein are
critical for diarrhea induction. The identification of critical residues makes
it a routine matter
for one skilled in the art to determine whether a given rotavirus is likely to
cause diarrhea by
comparing the amino acid sequence of the NSP4 protein to known sequences. For
example,
comparisons can be made using virulent and avirulent pairs of amino acid
sequences. In
addition, the identification of NSP4 sequences that correlate to an attenuated
phenotype
malces it a routine matter to construct attenuated reassortment viruses that
include such an
NSP4 sequence, using techniques that are well known to those skilled in the
art. This permits
the construction of rotaviruses for use as vaccines that retain the
antigenicity of the virulent
strain yet display an attenuated phenotype as a result of the incorporation
into the genome of
the virus a nucleic acid coding for an NSP4 protein having a selected
sequence.
Example 25
Preparation and use of an NSP4 toxoid
Vaccines comprising NSP4 in the form of a toxoid may be prepared from purified
NSP4 protein, NSP4 114-135, NSP4 120-147, NSP4 112-17S or NSP4 112-150. The
purified protein can be chemically treated, using known techniques, to
inactivate the
biological activity of the NSP4 protein while retaining the immunogenicity.
For example, the
purified protein may be treated with a 10% solution of formaldehyde at about
37, C for about
an hour. One skilled in the art will recognize that other equivalent protocols
to produce a
toxoid may be employed without deviating from the spirit of the invention.
After chemical
treatment the toxoid will typically be washed with buffer, for example
phosphate buffered
saline or the like, and formulated into a vaccine. The toxoid may be in solid
form such as
adsorbed to alum or the like. Alternatively, the toxoid may be in solution in
any
69
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
pharmaceutically acceptable liquid. The toxoid may be administered as a
vaccine in the
absence of adjuvant. A vaccine formulated with the toxoid may include
adjuvants including
but not limited to alum, Freund's complete and incomplete adjuvants, Ribi's
adjuvant,
bacterial and mycobacterial cell wall components and derivatives thereof,
liposomes and any
other adjuvant formulation known in the art. Vaccines thus formulated may be
administered
using parenteral or mucosal routes such as by intraperitoneal, intranasal,
intragastric,
subcutaneous, intramuscular, or rectal application.
Example 26
Characterization of the receptor for NSP4
The human intestinal cell line HT29 was assayed for sensitivity to NSP4. In
response
to purified NSP4, these cells showed an increase in intracellular calcium
levels. When these
cells are pre-treated with trypsin, the response is ablated. The binding of
radiolabeled NSP4
protein to responsive cells is dose-dependent and saturable as would be
expected for a
receptor dependent phenomenon. Taken together, these two results demonstrate
that NSP4
binds to a protein receptor. Recent tests with respiratory epithelial cells
have demonstrated
that these cells do not respond to NSP4 and do not bind radiolabeled NSP4
protein. It is well
within the ability of one of ordinary skill in the art to identify the
receptor by expression
cloning in these nonresponsive cells that do not bind NSP4. The mRNA from a
responsive
cell can be isolated using standard techniques and reverse transcribed into
cDNA. This
cDNA can then be inserted into a vector and then used to transform the
nonresponsive cell
line. One skilled in the art is cognizant that any vector may be used in this
invention.
Exemplary vectors include, but are not limited to, insect expression vectors,
bacteria
expression vectors, mammalian expression vectors or viral expression vectors.
Alternatively,
the genomic DNA from the responsive cells may be inserted into a vector and
used to
transform the nonresponsive cell line. The transformed cells will be screened
for the
expression of the receptor using routine techniques, for example, by screening
for cells
capable of binding radiolabeled NSP4. Cells that express the receptor will be
isolated.
Example 27
Preparation of SA 11 clone 3
Plaque-purified simian rotavirus SAll clone 3 (SA11, serotypes P3B[2],G3) was
cultivated in monkey kidney MA104 cells in the presence of trypsin. The 50%
diarrhea dose
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
(DD50) of the SAl l virus stock for Balb/C suckling mice was 1.4x104 PFU. A
stock of the
wild type murine rotavirus ECwt (P[16],G3) was prepared from the infected
intestines of
orally inoculated five-day-old mouse pups (Feng et al., 1994 and O'Neal et
al., 1997). The
titer of this ECwt stock in mouse pups was 2x10$ DD50 per ml.
Example 28
Preparation of SAll-NSP4112-175
NSP4 112-175 was produced in an insect cell suspension culture system and
purified
by immunoaffinity chromatography as reported elsewhere (Zhang et al., 2000).
The purified
NSP4 112-175 was lyophilized, and stored at 4 C in a dessicator until used.
SDS-15%
PAGE/silver staining and Western blot were used to evaluate protein purity.
Example 29
Preparation of SAll NSP4 and VP6
Full-length NSP4 production and purification were conducted in an insect cell
suspension system, purified by FPLC and immunoaffmity chromatography (Zhang et
al.,
1998 and Tian et al., 1996). VP6 was expressed in SF9 cells grown in optimized
serum-free
media, SF90011 SFM (Gibco, Grand Island, NY), acid purified by CsCI isopycnic
centrifugation (Zeng et al., 1996).
Example 30
Detection of serum antibodies by ELISA
All ELISAs were performed on 96-well polyvinyl chloride microtiter plates
(Dynatech, McLean, VA) (Ciarlet, et al., 1998 and Johansen, et al., 1999). To
detect the
mouse serum antibodies, the coating concentration of NSP4 was 24 ~,g/ml, NSP4
112-175
was 8 ~g/ml, and VP6 was 15 ~g/ml. Horseradish peroxidase-conjugated goat anti-
mouse 1g
(H+L) (Southern Biotechnology Associates, Inc., Birmingham, AL) was used to
detect the
bound mouse antibodies. The optimum dilution of antibody conjugates was
determined by
checkerboard titration. TMB peroxidase substrate, tetramethylbenzidine and
H202, was used
as chromogenic reagent (Kirkegaard and Perry Laboratories, Gaithersburg, MD).
Optical
densities (0.D.) at 450 mn were measured with an ICN Flow Titertech Multiscan
Plus MKl 1
plate reader (McLean VA). Antibody titers were defined as the reciprocal of
the highest
71
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
dilution giving a net O.D. value (0.D. value of detected serum minus O.D.
value of pooled
pre-immune serum) higher than 0.1.
Example 31
Plaque assay to detect infectious virus in the intestines of rotavirus SAll-
challenged
suckling mice
The level of replication of SAl l in the intestine of suckling mice was
determined by
titration of infectious virus by plaque assay (Ramig 1988). Similar
experiments could not be
performed with ECwt challenged mice because the ECwt virus does not replicate
efficiently
in vitro. Additional moclc-infected (PBS) mice were also included in this
experiment as
controls. The entire intestinal tract was removed from one SA11- and mock-
infected mouse
pup from 1-7 days post infection (DPI). The experiment was repeated once, for
a total of two
mouse pups for every timepoint. Each intestinal tract was homogenized
separately in 1 ml of
serum-free medium 199, extracted with an equal volume of Freon (1,1,2-
Trichloro-1,2,2-
trifluoroethanol, Fisher Scientific, Springfield, NJ), the water soluble phase
was collected and
treated with 20 ~.g/ml of trypsin for 30 min at 37 C. Each intestinal sample
was tested in
duplicate for virus titer in plaque assays with MA104 cells.
Example 32
Preparation of baculovirus recombinants
S.frugiperda insect cells (Sf~) were grown and maintained in TNM-FH (Hinks)
medium (Gibco, Grand Island, NY) containing 10% fetal bovine serum (FBS).
Baculovirus
recombinants encoding the following rotavirus proteins were used:
pFastBac/SA11-10 112-
175 [NSP4 112-175 of SAl l c1 3] (Zhang et al., 2000), pAc461/SAl l-10 [NSP4
of SAll c1
3] (Au et al., 1989), and pAc4461/SAl l-6 [VP6 of SA1 l c1 3] (Estes et al.,
1987).
Example 33
Immunization of dams with NSP4 as 114-175
NSP4 as 112-175 was expressed in and purified from the medium of insect Sf9
cells.
Purified NSP4 112-175 contained a single band with an apparent molecular
weight of 7,000
identified by SDS-15%p PAGE/silver staining (FIG. 11A, arrow). A single band
was also
visualized by Western blot using rabbit anti-NSP4 peptide 114-135 (FIG. 11B,
arrow), rabbit
anti-NSP4 full-length antiserum, or a rabbit anti-NSP4 peptide 120-147
antiserum.
72
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
Seronegative female mice were subcutaneously and intramuscularly immunized
with
14 ~.g NSP4 112-175 plus 20 ~,g Quillaja saponaria [adjuvant QS-21 (I~ensil,
et al., 1991)]
(NSP4-dam) or with 20 ~,g QS-21 alone (QS-21-dam). The first inoculation was
followed by
a booster two weeks later at which time breeding was begun. A second booster
was given on
the tenth day of breeding. Each dam was tail-bled on the indicated days for
determination of
antibodies by ELISA.
The control group (QS-21-dam) was inoculated with QS-21 alone. In the initial
experiments, pups were nursed by their own dams. The serum antibody titers in
NSP4-dams,
QS-21-dams and all pups were determined on 0, 7, and 16 DPP. The serum
antibody titers to
NSP4 aa112-175 and to NSP4 full-length detected by ELISA were essentially the
same
(FIG. 12A and FIG. 12B). The results show that (i) sera from NSP4-dams had
antibody
geometric mean titers (GMT) as high as 9x104 to both NSP4 aa112-175 and NSP4
full-
length; (ii) serum GMT were maintained throughout the experimental period in
the NSP4-
dams; (iii) NSP4-pups acquired only 3% of NSP4-dam titers transplacentally
(NSP4-pups on
0 DPP), but the majority of NSP4-specific antibody was obtained by lactogenic
transfer
(NSP4-pups on 7 and 16 DPP); (iv) the sera from QS-21-dams lacked antibody to
either
NSP4 1I2-175 or to NSP4 full-length; and (v) sera from the QS-21-pups lacked
detectable
antibody to NSP4, similar to their mothers, throughout the experimental
period.
Example 34
Cross-nursing of neonates induces changes in serologic antibody levels
Within 4 hrs of birth, neonatal pups bone to NSP4-dams and QS-21-dams were
immediately segregated from their biological mothers and cross-nursed by the
dams in the
other group.
Cross-nursing of neonates induces dramatic changes in serologic antibody
levels
against both NSP4 aa112-175 and full-length NSP4 in the pups. To compare the
importance
of transplacental and lactogenic transfers of antibody, cross-nursing
experiments were
performed. Pups were removed from their biological mothers within 4 hrs post
parturition
and cross-nursed. The results in FIG. 13A and 13B show that (a) sera from the
NSP4-pups
cross-nursed by QS-21-dams maintained their low antibody status (GMT, 3x103)
during the
experimental period, at levels similar to their antibody level at birth (white
bars); and (b) by 7
DPP, QS-2I-pups cross-nursed by NSP4-dams acquired antibody titers in the sera
(GMT,
73
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
9x104) similar to those of their nursing NSP4-dams which were maintained
throughout the
experimental period (gray bars). Cross-nursing experiments confirmed that 97%
of antibody
in pups was acquired from lactogenic transfer and only 3% from transplacental
transfer.
Example 35
Immunization of dams with NSP4 112-175 induces protection of suckling mice
against
SAll-induced diarrhea
At 7 DPP, each pup was challenged by stomach gavage with either 20 diarrhea
dose
50% (DD50) of SAl l or 10 DD50 of ECwt in 50 ~,1 of endotoxin-free PBS. Virus
inocula
were not trypsin-activated prior to inoculation. All cages were coded and
individual mice
were checked for diarrhea daily for 7 or 8 days after inoculation by gentle
palpation of their
abdomen. Stool classification was: 0, no stool; 1, normal stool; 2, normal
stool accompanied
with yellow pasty stool; 3, all yellow pasty stool; 4, milky-liquid stool. The
pups with a stool
score >_2 were considered to have diarrhea.
To evaluate whether antibody to NSP4 112-175 can mediate protection against
rotavirus-induced diarrhea, pups were challenged orally with 20 DD50 of SA11
at 7 DPP and
checked for diarrhea 0-7 DPI. All QS-21-pups developed diarrhea between 2-4
DPI which
generally lasted at least two days (Table 7). Only 50% of the NSP4-pups
developed diarrhea
and the duration of diarrhea was generally limited to one day. The percentage
of NSP4-pups
with diarrhea compared to QS-21-pups was significantly lower (P=0.007). The
mean
diarrhea scores of NSP4-pups were also lower (2.8) compared to QS-21-pups
(3.5).
74
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
O m N
'~ o
O
b
N O
o N o
O w
~ o II
P H
-I O
~ ~ d' M ,-y o N ~
~
+
.
y M ~~.ra
O '
~. o
Cet~ cd o N N O d'
EI 1 ~ M O N O ~ O N
~ II
~
., "L7O
a
N O '3W
\ o N
~
o
N v0 v p.,
v O
~, t~ O O
~
~ o ~
O 4~
~Q ~'
N
~
O D
i.-i
N ~
,--~ N ~ ~
W
~a
a
v~ ~ d' ,.~
V
U
y~.
r
~
~
a
~wZ
0
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
Example 36
Passively acquired antibody to simian rotavirus SAll NSP4112-175 protects pups
NSP4 112-175 was administered parenterally with the saponin adjuvant QS-21,
rather
than Freund's adjuvant, because QS-21 may be licensed for use in humans and is
more potent
than aluminum phosphate (Ciarlet et al., 1998).
It was tested whether passively acquired antibody to simian SA11 NSP4 112-175
could protect pups from a heterotypic challenge with 10 DD50 of virulent
marine rotavirus
ECwt. Similar to the results with homotypic virus challenge with SAl l, fewer
NSP4-pups
(44%) were not protected from ECwt- induced diarrhea, while 100% of QS-21-pups
developed diarrhea (P=0.02) (Table 8). The mean diarrhea scores in the NSP-4
pups (2.8)
were also lower compared to the QS-21-pups (3.3) (Table 8). In addition, the
onset of
diarrhea in NSP4-pups was later and was of shorter duration than in the QS-21-
pups. Thus,
NSP4 112-175 administered parentally induced protection of suckling mice
against diarrhea
induced by heterotypic marine ECwt rotavirus.
76
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
U
N -ii M 00
7 m N .,.~.,
C~
N
O O
O O ~ rUn
v dd~'~~
0~
~
O N
U
~~z
N ~
II
~ a~
o .-,
o
""
n.i ~ ~n O O U
Ci
V _
b o
" l~
~ ~ O O
, ~D O
I~
.~ 0
CC
U
~ W ,~ W W a a
~ a
,
;b o m m (~
t~ o
.~ ~ H a" O
,~ ~ N
n.i v~ oo c~
C~ a o o o .~1
~ O \ p
, '
~ N
~
W ' O
' '~O
~
'~ i~ 3
~ 0 0
0
~,,..~
d' v
+~
N
~
C) O U ' O a~
~ '
O
N N O O b0
N
"dO
'a'b
C~ . N
~
V7
~ I~ U p
_ U ~ _ _ '
N N
_ U
~ ~ N ,- d O
~ ~ U
'
O . U U
'
v, o ~
.d 3 a .~ ~ 3
z
a
77
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
Example 37
Antibody to NSP4 reduced virus replication
Rotavirus replication is reduced in pups delivered to and nursed by the dams
immunized with NSP4 112-175 plus QS-21. To investigate the possible effect of
passively
acquired antibody to NSP4 on rotavirus replication, the infectious virus
titers of SAl l in the
intestines of a subset of pups from NSP4-dams and QS-21-dams were determined
in two
separate experiments. No virus was detected from the intestines of either of
the mock-
infected control pups (FIG. 14A, FIG. 14B and FIG. 14C). The yields of
infectious SAll
were consistently higher and were detected for a longer period of time in the
two QS-21-
pups, compared to virus titers in the two NSP4-pups.
Example 38
Immunization of dams with NSP4 112-175 induces higher serologic antibody
titers
against SAll VP6
Dams were immunized with NSP4 aa112-175, both the dams and pups were orally
challenged with rotavirus SAl l .
This immunization induced higher serologic antibody titers against SAl l VP6
in both
dams and pups after pups were orally challenged with rotavirus SAll. It was
also
determined whether pre-existing antibody to NSP4 would alter the kinetics of
antibody
acquisition to rotavirus structural proteins by testing the serum antibody
titers to VP6. NSP4-
pups and QS-21-pups, but not dams, were orally challenged with 20 DD50 SAll.
Antibody
to VP6 was not detectable in any of the dams and pups at 0 DPI, but was
detectable at 12 DPI
(FIG. 15). The serum antibody titers to VP6 in the NSP4-pups (GMT, 3x103) were
significantly higher than those in the QS-21-pups (GMT, 3x102) (P<0.001). The
serum
antibody titers to VP6 in the NSP4-dams (GMT, 8x103) were also significantly
higher than
those in the QS-21-dams (GMT, 2x103) (p=0.005). These results indicated that
(i) not
unexpectedly, infectious rotavirus was transmitted from the inoculated pups to
their
unirioculated dams; (ii) pre-existing actively acquired antibody to NSP4 in
the dams
enhanced the immune response to VP6, a non-related immunogen.
78
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
Example 39
Cross-nursed pups show protection from lactogenic antibody
In a separate cross-nursing and challenge experiment (Table 9), nine QS-21-
pups
(group A) and 10 NSP4-pups (group B) were nursed by their biological mothers
during the
experimental period. Within 4 hrs of birth, 13 QS-21-pups were transferred to
and cross-
nursed by NSP4-dams (group C), and 15 NSP4-pups were transferred to and cross-
nursed by
QS-21-dams (group D) during the experimental period. All the pups were orally
challenged
with 20 DD50 SAll. As observed in the initial challenge experiment (Table 8),
all of the
QS-21-pups in group A developed diarrhea and only 50% of the NSP4-pups in
group B
developed diarrhea (P=0.026) and the mean diarrhea scores in group B (2.6)
were lower than
group A (3.3). Only 46% of the QS-21-pups cross-nursed by NSP4-dams (group C)
developed diarrhea, their mean diarrhea score was 2.8 and they were
significantly protected
against SA11 diarrhea (p=0.010) compared to QS-21-pups (group A). The onset of
diarrhea
in NSP4-pups cross-nursed by QS-21-dams (group D) was delayed; the percentage
of pups
with diarrhea was significantly different at 2 dpi compared to QS-21-pups in
group A (0% vs.
335 of diarrhea, P=0.041). The NSP4-pups cross-nursed by QS-21-dams (group D)
had a
lower diarrhea score (2.9) and showed partial protection (27%) against
diarrhea relative to the
QS-21-pups in group A, but protection was not siguficantly higher (p=0.090).
79
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
a~
c~a ~ o M ~0 00
a~
M N N
,fl a _
,Q.i H \ ~ o O N o M ~ o _~ v
P..W~ O ~ O ~ ~ ~ v ~ M
OOv1 ~~O .d.~O
H
U
. ~ ~~ '~ _
o O~ o~ ~ ~ o~ ,M-, C/~ o ~ C/~
\O ~ O w ~ W~ w
V ~ o N
.~ ..vi a~
_ b
.~I p ~ ° O~1 0 o O o ,..M_, O o ~ ~ N
~r U b O O w 01 w M
O ~ .~. ~ Q M
a\ '~ ~' . ~ P~ P-~
b O P,
i~ ~ ~ ~ ~
"~.' ~ ~ N o ~ o ~ C/~ o ,-Mi O o ~ O ~ S3,
ooz QaQ Qao ~ o
'"' '.G ~ ° o
en
~ ~ LV ~, N N _N N ~., [n ~'' +.;
~,' ~ ~ N c~d a' c~d a
a ~~ '~~ a o
a~
U . ~ ~ r~ ~ P..~ ~, p.,
z z o
'~ w
U . ~ .~ N r' C/~ N '-' V~ v ' ' p
y~ ~ ~ ~a ~ ~a
a ~~ a
'
z z
0. U ~1 ~ ~wZ
~i~~ v
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
Example 40
NSP4-VP2/VP6 construct
A VP2-NSP4 fusion protein was constructed such that the coding region of gene
10,
amino acids 112-175 without the stop codon, was cloned at the 3'-end of gene
2, amino acids
94-881 (SEQ.ID.N0:39 or SEQ.ID.N0:37). A linker composed of 3 alanine residues
followed by one alanine and one serine residues were included between the two
coding
regions. This construct was cloned behind the p10 promoter of the baculovirus
transfer
vector pBAC4x-1. The entire coding region for VP6 (SEQ.ID.N0:38) was cloned
behind the
polyhedrin promoter in the pBAC4x-1 vector containing VP2-NSP4 fusion gene.
Following the standard co-transfection and recombinant baculovirus isolation,
two
baculovirus recombinants were obtained. Virus-like particles (VLPs) were
expressed,
purified and analyzed by Western blot and electron microscopy. VP2-NSP4 and
VP6 were
detected by Western blot analysis using a polyclonal antibody against SA11
virus (FIG. 16).
The presence of VP2-NSP4 was confirmed using an antiserum against NSP4. The
liner
used above is one possibility of making such a fusion protein with VP2.
One skilled in the art realizes that the fusion protein can contain one or
more than one
copy of a NSP4 protein. A similar method could be used to make VLPs carrying
proteins or
peptides from other pathogens.
Example 42
In vitro Assay for NSP4120-147
Antiserum to the NSP4 peptide from the rotavirus strain SA11 reacts with NSP4
from
at least one other strain, the porcine rotavirus strain OSU that is classified
in a separate
genetic' group from the SAl l NSP4. An if2 vitro assay was developed to
measure "biologic
neutralizing activity" of antisera to regions of NSP4 that would block
enterotoxin or signaling
of the enterotoxin. First, NSP4 was tested to determine whether it can affect
the
transepithelial resistance (TER) of polarized epithelial cells grown on
filters. It was found
that polarized epithelial cells displayed high resistance and this resistance
begins to decrease
about 16 hours after the cells were treated with NSP4. Next, antibodies to
different regions
of NSP4 were added to examined if this drop in TER can be prevented "or
neutralized".
Antibodies to the NSP4 2-22 peptide, NSP4 114-135 peptide, NSP4 120-147
peptide, and to
the full-length protein were tested. Preimmune serum for these peptides was
also tested.
81
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
These results show that pre-immune serum and antiserum to the 2-22 peptide do
not block the
drop in TER in cells treated with NSP4. The lack of neutralization by the
antibody to 2-22 is
consistent with the results that this peptide does not cause diarrhea in mice
and immunization
of mice with the peptide does not result in protective immuiuty. In contrast,
antibodies to
NSP4 114-135, NSP4 120-147 and to the full-length protein blocked the drop in
TER in cells
treated with NSP4 (FIG. 17 and Table 10). Thus, these data indicate that
antibodies to NSP4
120-147 behave in a manner similarly to antibodies to NSP4 114-135. Since the
114-135
peptide causes diarrhea in mice and antibodies to NSP4 114-135 have also been
shown to
prevent diarrhea in mice, and the NSP4 120-147 peptide causes diarrhea in
mice, these ih
vitro" neutralization data" support the claim that immunization with the
peptide NSP4 120-
147 will result in protection from diarrhea. This data demonstrates that if a
peptide causes
diarrhea in mice, antibody to that peptide will protect against diarrhea. The
data indicate that
the diarrhea is caused by activation by the peptide (or protein) of a cell
signaling pathway that
results in diarrhea. Antibody to these peptides likely stops this signaling
process.
82
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
F4
z
0
~ z z z ~ z
0
0 z
~..~~
~z
~
N
c~
a~
cd
~U
0
0
z o
z
z ~
w
o ~ ~ ~ ~ ~ ~ ~
~
, , .
~ ~
N
c~ d'd- d' d' d' d' d'
~ ~ ~ ~ ~
~
~ ~ ~ ~ r~ C ~
- ~/ ' /~ ~
'
c V C c ~ d l
111d 1 /~d zM z~ z~-
z z~ z z~,
~
,~.~ ,~ ,~ ,.~ ,.~ ~
' Z N Z ~'. Z Cj
~ ~A ~A ~A ~A WA b0
A , ,- N
~ N N ~ N N N
~ N ~ ~
_ _ _ _ _ _
,~r',
~ ~ ~
H w r~. wZ w. wZ w. r~Z
83
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
All patents and publications mentioned in the specification are indicative of
the level
of those skilled in the art to which the invention pertains. All patents and
publications are
herein incorporated by reference to the same extent as if each individual
publication was
specifically and individually indicated to be incorporated by reference.
REFERENCES
A.P. Morns S.A. Cunningham, A. Tousson, D.J. Benos, R.A. Frizzell, Am J.
Physiol.
266, C254 (1994).
A.Z. Kapikian and R.M. Chanock, in Virology, B.N. Fields and D.M. Knipe, Eds.
(Raven Press, New York, 1996) chap. 55.
American Academy of Pediatrics. 1998. Prevention of rotavirus disease:
Guidelines
for use of rotavirus vaccine. Pediatf°ics 102:1483-1491.
Au, K. S., Chen, W. K., Burns, J. W., and Estes, M. K. 1989. J. Yi~ol. 63:4553-
4562.
B.R. Grubb, Ain. J. Physiol. 268, 6505 (1995).
Ball, J. M., Tian, P., Zeng, C.Q.Y., Morris, A. P., and Estes, M. K. 1996.
Science
272:101-104.
Bern, C., Marlines, J., de Zoysa, L, and Glass, R. I. 1992. The magnitude of
the
global problem of diarrhoeal disease: a ten-year update. Bull. Wof-ld health
O~gaya. 70:705-
714. '
C.L. Sears, R.L. Guerrant, J.B. Kaper, in Infections of the Gastrointestinal
Tract, M.J.
Blaser, P.D. Smith, J.I. Ravdin, H.B. Greenberg, R.L. Guerrant, Eds. (Raven
Press, New
York, 1995), chap. 44;
C.Q.-Y. Zeng, M.J. Wentz, J. Cohen, M.K. Estes, R.F. Ramig, J. Virol., in
press
(1996).
CDC MMWR. 1999. Intussusception among recipients of rotavirus vaccine-United
States. 1998-1999. Morb. Mortal. WKLY. Rep. 48:577-581.
Ciarlet, M., and Conner, M. E. 2000. In J. Gray and U. Desselberger (ed.),
Methods in
Molecular Medicine. Humana Press Inc., New Yorle.
84
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
Ciarlet, M., Crawford, S. E., Barone, C., Bertolotti-Ciarlet, A., Ramig, R.
F., Estes,
M. K., A~ CONNER, M. E. 1998. J. Viol. 72:9233-9246.
Ciarlet, M., Lipxandi, F., Corner, M. E., and Estes, M. K. 2000. AYCh. Virol.
145:371-
383.
Cunliffe, N. A., Woods, P. A., Leite, J. P., Das, B. K., Ramachandran, M.,
Bhan,
M. K., Hart, C. A., Glass, R. L, and Gentsch, J. R. 1997. J. Med. Virol. 53:41-
50.
D.Y. Graham, J.W. Sackman, M.K. Estes, Dig. Dis. Sci. 29, 1028 (1984).
Desselberger, U., and Estes, M. K. 2000. In J. Gray and U. Desselberger (ed.),
Methods in Molecular Medicine. Humana Press Inc., New York. p 239-258.
Dong, Y. J., Zeng, C. Q. Y., Ball, J. M., Estes, M. K., and Morris, A. P.
1997. Proc.
Natl. Acad. Sci. U.S.A. 94:3960-3965.
Dyson, et al. Ann. Rev. Biophys. Biophysical Chem.l7, 305 (1988).
Dyson, et al., Biochemistry, 31, 1458 (1992)
Dyson, et al., Biophysical Chem. 20, 519 (1991).
Dyson, et al., FASEB J. 9, 37 (1995).
Dyson, et al., J. Mol. Biol. 201, 161 (1988).
Dyson, et al., J. Mol. Biol. 201, 201 (1988).
E. Kaiser, R.L. Colescott, C.D. Bosinger, P.I. Cook, Anal. Biochem. 34 595
(1970).
Estes, M. K. 1996. Rotavirus and their replication, p 1625-1655. Ih B. N.
Fields,
D. M. Knipe (ed.), Virology 3'~a ed. Raven Press, New York.
Estes, M. K., Crawford, S. E., Penaranda, M. E., Petrie, B. L., Burns, J. W.,
Chan, W.
K., Ericson, B. L., Smith, G. E., and Summers, M. D. 1987. J. ViYOI. 61:1488-
1494.
Evans, D. G., Evans, D. J. Jr., Opekun, A. R., and Graham, D. Y. 1988. FEMS
MicYObiol. In2muh.ol. 1:117-125.
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
Feng, N., Burns, J. W., Bracy, L., and Greenberg, H. B. 1994. J. Tli~ol.
68:7766-7773.
G.P. Johnson et al., Anal. Chem. 58, 1084 (1986).
G.W. Both, L.J. Siegman, R.R. Bellamy, P.H. Atkinson, J. Virol. 48, 335
(1983).
H. Holzel, D.W. Cubitt, D.A. McSwiggan, P.J. Sanderson, J. Church, J. Infect.
Dis. 2,
33 (1980).
H. Margolit et al., J. Immunol. 138, 2213 (1987).
H.B. Greenberg, H.F. Clark, P.A. Offit, Curr. Top. Microbiol. Tmmunol. 185,
255.
H.B. Greenberg, H.F. Clark, P.A. Offit, ibid. 185, 255 (1994).
Horie, Y., Nakagomi, O., Koslumura, Y., Nalcagomi, T., Suzuki, Y., Oka, T.,
Sasaki,
S., Matsuda, Y., acid Watanabe, S. 1999. Yi~ology 262:398-407.
J. Collins, et al., J. Pediatr. Gastroenterol. Nutr. 7, 264 (1988).
J. Halvorsrud and I. Orstavik, Scand. J. Infect. Dis. 12, 161 (1980).
J. Levin and F.B. Bang, Throm. Diath. Haemorrh. 19, 186 (1968);
J. Yao, et al., J. Mol. Biol. 243, 736 (1994).
J.L. Wolf, G. Culcor, N.R. Ballcklow, R. Dambrauskas, J.S. Trier, Infec.
Ixnmun. 33,
565 (1981).
J.L. Wolf, G. Cukor, N.R. Ballcklow, R. Dambrauskas, J.S. Trier, Infec. Immun.
33,
565 (1981)
J.M. Ball, N.L. Henry, R.C. Montelaro, J.J. Newman, J. Immunol Methods 171, 37
(1994).
J.M.R. Parker, D.Guo, R.S. Hodges, Biochemistry 25, 5425 (1986).
J.P. McAdaragh et al., ibid 41, 1572 (I980);
J.W. Burns, et al., Virol. 207, 143 (1995).
86
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
Johansen, K., Hinkula, J., Espinoza, F., Levi, M., Zeng, C. Q. Y., Ruden, U.,
Vesikari,
T., Estes, M. K., and Svensson, L. 1999. J. Med. T~i~ol. 59:369-377.
K.W. Theil, E. Bohl, R. Cross, E. Kohler, A. Agnes, Am. J. Vet. Res. 39, 213
(1978);
Kapikian, A. Z., and Chanock, R. M. 1996. Rotavirus. W Vif°ology 3Y~
ed. B. N.
Fields, D. M. Knipe, editors. Raven Press, New York. 1657=1708.
Kensil, C. R., Patel, U., Lennick, M., and Marciani, D. J. 1991. J. Tmmunol.
146:431-
437.
Kirkwood, C. D., and Palombo, E. A. 1997. Trirology 236:258-265.
L.A. Carpino and G.H. Han, J. Org. Chem. 37, 5748 (1970).
L.J. Saif, L.A. Ward, L. Yuan, B.I. Rosen, T.L. To, in Proceedings of the
Sapparo
International Symposiums on Viral Gastroentritis, S. Chiba, S. Nakata, and
M.K. Estes, Eds.
(Arch. of Virol. Special Issue) in press; C.A. Mebus, Am. J. Dig. Dis. 21, 592
(1976).
L.M. Little and J.A. Shadduck, Infect. Immun. 38, 755 (1982);
L.R. Forte, et al., Am. J. Phys. 263, 0607 (1992).
M. Field, L.H. Graf, W.J. Larid, P.L. Smith, Proc. Natl. Acad. Science USA 75,
2800
1978);
M.G. Currie et al., Proc. Natl. Acad. Science USA 89, 947 (1992);
M.K. Estes, E.L. Palmer, J.F. Obijeski, Curr. Top. Microbiol. Tmmunol. 105,
123
(1983);
M.N. Burges, et al., Infect. Immunol. 221, 526 (1978);
M.P. Osborne et al., J. Pediatr. Gastroenterol. Nutr. 7, 236 (1988).
Matsui, S. M., Offit, P. A., Vo, P. T., Mackow, E. R., Benfield, D. A., Shaw,
R. D.,
Padilla-Noriega, L., and Greenberg, H. B. 1989. J. Clin. Microbiol. 27:780-
782.
Meyer, J. C., Bergmann, C. C., and Bellamy, A. R. 1989.
87
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
Morris, A. P., Scott, J. K., Ball, J. M., Zeng, C. Q. Y., O'Neal, W. K., and
Estes,
M. K. 1999. Am. J. Physiol.; GI 277:6431-6444.
N. Feng, H.W. Burns, L. Bracy, H.B. Greenberg, J. Virol. 68, 7766 (1994);
O'Neal, C. M., Crawford, S. E., Estes, M. K., and Conner, M. E. 1997. J.
Tlif°ol.
71:8707-8717.
Offit, P. A., and Clark, H. F. 1985. J. Infect. Dis. 152:1152-1158.
Offit, P. A., and Clark, H. F. 1985. J. Yi~ol. 54:58-64.
P. Tian, M.K. Estes, Y. Hu., J.M. Ball, C.Q.-Y. Zeng, W.P. Schilling, J.
Virol. 69, 576
(1995).
P. Tian, Y. Hu, W.P. Schilling, D.A. Lindsay, J. Eiden, M.K. Estes, J. Virol.
68, 51
(1994).
P.E. Wright, et al., Biochemistry 27, 7167 (1988).
P.Y. Chou and G.D. Fassman, Adv. in Enz. 47, 45 (1978).
Prasad, B. V. V., and Estes, M. K. 1997. hz W: Chiu, R. M. Burnett, and R. L.
Garcea (ed.), Structural Biology of Viruses. Oxford University Press, New
York, Oxford.
p 239-238.
Prasad, B. V. V., Rothnagel, R., Zeng, C.Q.Y., Jalcana, J., Lawton, J. A.,
Chiu, W.,
and Estes, M. K. 1996. Nature (Lohdoh) 382:471-473.
R.A. Giannella, Ann. Rev. Med. 32, 341 (1981);
R.A. Giannella, M. Luttrell, M. Thompson, Am. J. Phys. 245, 6492 (1983);
R.F. Raanig, Microbial Pathogenesis 4, 189 (1988).
R.L. Ward, M.M. McNeal, J.F. Sheridan, J. Virol. 64, 5070 (1990);
Ramig, R. F., 1988. Micy°ob. Pathog. 4:189-202.
Reichlin, Methods in Enzyn. 70, 159 (1980).
88
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
Riepenhoff Talty, P.C. Lee, P.J. Carmody, H.J. Barrett, P.L. Ogra, Proc. Soc.
Exp.
Biol. Med. 170, 146 (1982).
Riepenhoff Talty, P.C. Lee, P.J. Carmody, H.J. Barrett, P.L. Ogra, Proc. Soc.
Exp.
Biol. Med. 170, 146 (1982).
Ryan, E. T., Butterton, J. R., Smith, R. N., Carroll, P. A., Crean, T. L, and
Calderwood, S. B. 1997. hafect. Immun. 65:2941-2949.
T.J. Novitsky, Oceanus 27, 13 (1984).
Tian, P., Ball, J. M., Zeng, C. Q. Y., and Estes, M. K. 1996. J. Yisrol.
70:6973-6981.
Tian, P., Estes, M. K., Hu, Y., Ball, J. M., Zeng, C. Q. Y., and Schilling, W.
P. 1995.
J. Viol. 69:5763-5772.
W.G. Starkey et al., J. Gen. Virol. 67, 2625 (1986).
W.J. Krause, R.H. Freeman, L.R. Forte, Cell and Tissue Res. 260, 387 (1990).
Waltho, et al., Biochemistry, 32, 6337 (1993).
Woody, M. A., Krakauer, T., and Stiles, B. G. 1997. T~acci~ae 15:133-139.
X. Jiang, D.Y. Graham, P. Madore, T. Tanaka, M.K. Estes, Science 250, 1580
(1990).
Zent, C. Q. Y., Wentz, M. J., Cohen, J., Estes, M. K., and Ramig, R. F. 1996.
J.
Viol. 70:2736-2742.
Zhang, M. D., Zeng, C. Q. Y., Morris, A. P., and Estes, M. K. 2000. A
functional
NSP4 enterotoxin peptide secreted from rotavirus-infected cells. Submitted for
publication.
Zhang, M., Zeng, C. Q. Y., Dong, Y., Ball, J. M., Sarif, L. J., Morris, A. P.,
and Estes,
M. K. 1998. J. Virol. 72:3666-3672.
One of skill in the art readily appreciates that the present invention is well
adapted to
carry out the objectives and obtain the ends and advantages mentioned as well
as those
inherent therein. Vaccines, vectors, methods, procedures and techniques
described herein are
presently representative of the preferred embodiments and are intended to be
exemplary and
89
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
are not intended as limitations of the scope. Changes therein and other uses
will occur to
those skilled in the art which are encompassed within the spirit of the
invention or defined by
the scope of the pending claims.
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
SEQUENCE LISTING
<110> ESTES, MARY
<120> ROTAVIRUS ENTEROTOXIN NSP4 AND METHODS OF USING SAME
<130> P01932W01
<140> TBA
<141> November 2, 2001
<150> 09/705,621
<151> November 3, 2000
<160> 39
<l70> PatentIn version 3.0
<210> l
<211> 22
<212> PRT
<213> Rotavirus NSP4 114-135
<400> 1
Asp Lys Leu Thr Thr Arg Glu Ile Glu Gln Val Glu Leu Leu Lys Arg
1 5 l0 15
Ile Tyr Asp Lys Leu Thr
<210> 2
<211> 21
<212> PRT
<213> Rotavirus NSP4 2-22
<400> 2
Glu Lys Leu Thr Asp Leu Asn Tyr Thr Leu Ser Val Ile Thr Leu Met
1 5 10 15
Asn Asn Thr Leu His
<210> 3
<211> 33
<212> PRT
<213> Rotavirus NSP4 90-123
<400> 3
Thr Lys Asp Glu Ile Glu Lys Gln Met Asp Arg Val Val Lys Glu Met
1 5 10 15
Arg Arg Gln Leu Glu Met Ile Asp Lys Leu Thr Thr Arg Glu Ile Glu
20 25 30
Gln
-1-
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
<210> 4
<211> 22
<212> PRT
<213> Mutated Rotavirus NSP4 114-135 Peptide
<400> 4
Asp Lys Leu Thr Thr Arg Glu Ile Glu Gln Val Glu Leu Leu Lys Arg
1 5 10 15
Ile Lys Asp Lys Leu Thr
<210> 5
<211> 20
<212> PRT
<213> Rotavirus NV 464-483
<400> 5
Asp Thr Gly Arg Asn Leu Gly Glu Phe Lys Ala Tyr Pro Asp Gly Phe
l 5 10 15
Leu Thr Cys Val
<210> 6
<211> 28
<212> PRT
<213> Rotavirus NSP4 120-147
<400> 6
Glu Ile Glu Gln Val Glu Leu Leu Lys Arg Ile Tyr Asp Lys Leu Thr
1 5 10 15
Val Gln Thr Thr Gly Glu Ile Asp Met Thr Lys Glu
20 25
<210> 7
<211> 175
<212> PRT
<213> Porcine Rotavirus
<400> 7
Met Ala Leu Leu Ala Ala Leu Ala Thr Thr Leu Ser Val Ile Thr Leu
1 5 10 15
Met Ala Ala Thr Leu His Ser Ile Ile Gly Ala Pro Gly Met Ala Thr
20 25 30
Pro Pro Thr Ile Ala Ser Val Leu Thr Val Leu Pro Thr Leu His Leu
35 40 45
Ala Ser Ile Pro Thr Met Leu Ile Ala Leu Leu Thr Ser Leu Cys Ser
50 55 60
Thr Leu Val Ile Leu Thr Cys Met Val Thr Ile Ile Ala Thr Leu Leu
65 70 75 80
-2-
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
Leu Leu Ala Gly Thr Leu Gly Gly Val Thr Thr Leu Ala Gly Ile Gly
85 90 95
Gly Gly Met Ala Ala Ile Ile Leu Gly Met Ala Ala Gly Leu Gly Met
100 l05 ll0
Ile Ala Leu Leu Thr Thr Ala Gly Ile Gly Gly Val Gly Leu Leu Leu
115 120 125
Ala Ile His Ala Leu Leu Ala Ala Ala Pro Va1 Ala Ala Ile A1a Met
130 135 140
Ser Leu Gly Pro Ala Gly Leu Ala Ile Ala Thr Leu Ala Gly Thr Gly
145 150 155 160
Ser Gly Leu Ala Pro Thr Gly Pro Ser Gly Val Thr Ala Ser Met
165 170 175
<210> 8
<21l> l75
<212> PRT
<213> OSU-a
<400> 8
Met Asp Lys Leu Ala Asp Leu Asn Tyr Thr Leu Ser Val Ile Thr Leu
1 5 10 15
Met Asn Asp Thr Leu His Ser Tle Ile Gln Asp Pro Gly Met Ala Tyr
20 25 ~ 30
Phe Pro Tyr Ile Ala Ser Val Leu Thr Val Leu Phe Thr Leu His Lys
35 40 45
Ala Ser Ile Pro Thr Met Lys Ile Ala Leu Lys Thr Ser Lys Cys Ser
50 55 60
Tyr Lys Val Ile Lys Tyr Cys Met Val Thr Tle Ile Asn Thr Leu Leu
65 70 75 80
Lys Leu Ala Gly Tyr Lys Glu Gln Val Thr Thr Lys Asp Glu Ile Glu
85 90 95
Gln Gln Met Asp Arg Ile Ile Lys Glu Met Arg Arg Gln Leu Glu Met
100 l05 l10
Ile Asp Lys Leu Thr Thr Arg Glu Ile Glu Gln Val Glu Leu Leu Lys
115 120 125
Arg Ile His Asp Lys Leu Ala Ala Arg Ser Val Asp Ala Ile Asp Met
130 135 l40
Ser Lys Glu Phe Asn Gln Lys Asn Ile Arg Thr Leu Asp Glu Trp Glu
145 150 155 160
Ser Gly Lys Asn Pro Tyr Glu Pro Ser Glu Val Thr Ala Ser Met
165 170 175
<210> 9
<211> 63
-3-
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
<212> PRT
<213> Rotavirus NSP4 112-175
<400> 9
Met Ile Asp Lys Leu Thr Thr Arg Glu Ile Glu Gln Val Glu Leu Leu
1 5 10 15
Lys Arg Ile Tyr Asp Lys Leu Thr Val Gln Thr Thr Gly Glu Ile Asp
20 25 30
Met Thr Lys Glu Ile Asn Gln Lys Asn Val Arg Thr Leu Glu Glu Trp
35 40 45
Glu Ser Gly Lys Asn Pro Glu Pro Lys Glu Val Thr Ala Ala Met
50 55 60
<210> 10
<2l1> 39
<212> PRT
<213> Rotavirus NSP4 112-150
<400> 10
Met Ile Asp Lys Leu Thr Thr Arg Glu Ile Glu Gln Val Glu Leu Leu
1 5 10 15
Lys Arg Ile Tyr Asp Lys Leu Thr Val Gln Thr Thr Gly Glu Ile Asp
20 25 30
Met Thr Lys Glu Ile Asn Gln
<210> 11
<211> l75
<212> PRT
<213> Simian 11 Rotavirus (strain SA11)
<400> 11
Met Gly Leu Leu Thr Ala Leu Ala Thr Thr Leu Ser Val Ile Thr Leu
1 5 10 15
Met Ala Ala Thr Leu His Thr Ile Leu Gly Ala Pro Gly Met Ala Thr
20 . 25 30
Pro Pro Thr Ile Ala Ser Val Leu Thr Val Leu Pro Ala Leu His Leu
35 40 45
Ala Ser Ile Pro Thr Met Leu Ile Ala Leu Leu Thr Ser Leu Cys Ser
50 55 60
a
Thr Leu Val Val Leu Thr Cys Tle Val Thr Ile Pro Ala Thr Leu Leu
65 70 75 80
Leu Leu A1a Gly Thr Leu Gly G1y Ile Thr Thr Leu Ala Gly Ile Gly
85 90 95
Leu Gly Met Ala Ala Val Val Leu Gly Met Ala Ala Gly Leu Gly Met
100 105 110
-4-
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
Ile Ala Leu Leu Thr Thr Ala Gly Ile Gly Gly Val Gly Leu Leu Leu
115 120 125
Ala Ile Thr Ala Leu Leu Thr Val Gly Thr Thr Gly Gly Ile Ala Met
130 135 140
Thr Leu Gly Ile Ala Gly Leu Ala Val Ala Thr Leu Gly Gly Thr G1y
145 150 155 160
Ser Gly Leu Ala Pro Thr Gly Pro Ala Gly Val Thr Ala Ala Met
165 170 175
<210> 12
<211> 176
<212> PRT
<213> Rotavirus NSP4 SA 11 clone 3
<400> 12
Met Glu Lys Leu Thr Asp Leu Asn Tyr Thr Leu Ser Val Ile Thr Leu
1 5 10 15
Met Asn Asn Thr Leu His Thr Ile Leu Glu Asp Pro Gly Met Ala Tyr
20 25 30
Phe Pro Tyr Ile Ala Ser Val Leu Thr Val Leu Phe Ala Leu His Lys
35 40 45
Ala Ser Ile Pro Thr Met Lys Ile Ala Leu Lys Thr Ser Lys Cys Ser
50 55 60
Tyr Lys Gly Val Val Lys Tyr Cys Ile Val Thr Ile Phe Asn Thr Leu
65 70 75 80
Leu Lys Leu Ala G1y Tyr Lys Glu Gln I1e Thr Thr Lys Asp Glu Ile
85 . 90 95
Glu Lys Gln Met Asp Arg Val Val Lys Glu Met Arg Arg Gln Leu Glu
100 105 110
Met Ile Asp Lys Leu Thr Thr Arg Glu Tle G1u Gln Val Glu Leu Leu
115 120 125
Lys Arg Ile Tyr Asp Lys Leu Thr Val Gln Thr Thr G1y Glu Ile Asp
130 135 140
Met Thr Lys Glu Ile Asn Gln Lys Asn Val Arg Thr Leu Glu Glu Trp
145 150 155 160
Glu Ser Gly Lys Asn Pro Tyr G1u Pro Arg Glu Val Thr Ala Ala Met
165 170 175
<210> 13
<211> 175
<212> PRT -
<213> Rotavirus
<400> 13
Met Glu Lys Leu Ala Asp Leu Asn Tyr Thr Leu Gly Val Ile Thr Leu
1 5 10 15
-5-
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
Met Asn Asp Thr Leu His Asn Ile Leu Glu Glu Pro Gly Met Val Tyr
20 25 30
Phe Pro Tyr I1e Ala Ser Ala Leu Thr Val Leu Phe Thr Met His Lys
35 40 45
Ala Ser Leu Pro Ala Met Lys Leu Ala Met Arg Thr Ser Gln Cys Ser
50 55 60
Tyr Arg Ile Ile Lys Arg Val Va1 Va1 Thr Leu Val Asn Thr Leu Leu
65 70 75 80
Arg Leu Gly Gly Tyr Asn Asp Tyr Leu Thr Asp Lys Asp Glu Thr Glu
85 90 95
Lys Gln Ile Asn Arg Val Val Lys Glu Leu Arg Gln Gln Leu Ala Met
100 105 110
Ile Glu Lys Leu Thr Thr Arg Glu Ile Glu Gln Val Glu Leu Leu Lys
115 120 125
Arg Ile Tyr Asp Met Met Val Val Cys Arg Asp Arg G1u Ile Asp Met
130 135 140
Ser Lys Glu Thr Asn Arg Lys Ala Phe Lys Thr Leu His Asp Trp Gly
l45 150 155 160
Ser Asp Arg Asn Tyr Asp Asp Asn Thr Asp Val Ile Ala Pro Leu
165 170 175
<210> 14
<211> 175
<212> PRT
<213> GOTT-V
<400> 14
Met Asp Lys Leu Ala Asp Leu Asn Tyr Thr Leu Asn Val Ile Thr Leu
1 5 10 15
Met Asn Asp Thr Leu His Ser Ile Ile Gln Asp Pro Gly Met Ala Tyr
20 25 30
Phe Pro Tyr Ile A1a Ser Val Leu Thr Val Leu Phe Thr Leu His Lys
35 40 45
Ala Ser Ile Pro Thr Met Lys Ile Ala Leu Lys Thr Ser Lys Cys Ser
50 55 60
Tyr Lys Val Ile Lys Tyr Cys Met Va1 Thr Ile Ile Asn Thr.Leu Leu
65 70 75 80
Lys Leu Ala Gly Tyr Lys Glu Gln Val Thr Thr Lys Asp Gly Ile Glu
85 90 95
Gln Gln Met Asp Arg Ile Ile Lys Glu Met Arg Arg Gln Leu Glu Met
100 105 110
Ile Asp Lys Leu Thr Thr Arg Glu Tle Glu Gln Val Glu Leu Leu Lys
115 120 125
-6-
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
Arg Ile His Asp Lys Leu Val Ala Arg Pro Val Asp Ala Ile Asp Met
130 135 140
Ser Lys Glu Phe Asn Gln Lys Asn Ile Arg Thr Leu Asp Glu Trp Glu
145 150 155 160
Ser Gly Lys Asn Pro Tyr Glu Pro Ser Glu Val Thr Ala Ser Met
165 170 175
<210> 15
<211> 175
<212> PRT
<213> GOTT-A
<400> 15
Met Asp Lys Leu Ala Asp Leu Asn Tyr Thr Leu Ser Val Ile Thr Leu
1 5 ZO 15
Met Asn Asp Thr Leu His Ser Ile Ile Gln Asp Pro Gly Met Ala Tyr
20 25 30
Phe Pro Tyr Ile Ala Ser Val Leu Thr Val Leu Phe Thr Leu His Lys
35 40 45
Ala Ser Ile Pro Thr Met Lys Ile Ala Leu Lys Thr Ser Lys Cys Ser
50 55 60
Tyr Lys Val Ile Lys Tyr Cys Met Val Thr Ile Ile Asn Thr Leu Leu
65 70 75 80
Lys Leu Ala Gly Tyr Lys Glu Gln Val Thr Thr Lys Asp G1u Ile Glu
85 90 95
Gln Gln Met Asp Arg Ile Ile Lys Glu Met Arg Arg Gln Leu Glu Met
100 105 110
Ile Asp Lys Leu Thr Thr Arg Glu Ile Glu Gln Val Glu Leu Leu Lys
115 120 125
Arg Ile His Asp Lys Leu Ala Ala Arg Ser Val Asp Ala Ile Asp Met
130 135 140
Ser Lys Glu Phe Asn Gln Lys Asn Ile Arg Thr Leu Asp Glu Trp Glu
145 150 155 160
Ser Gly Lys Asn Pro Tyr Glu Pro Ser Glu Val Thr Ala Ser Met
165 170 175
<210> 16
<211> 751
<212> DNA
<213> Rotavirus NSP4 SA 11 clone 3
<400> 16
ggcttttaaa agttctgttc cgagagagcg cgtgcggaaa gatggaaaag cttaccgacc 60
tcaattatac attgagtgta atcactctaa tgaacaatac attgcacaca atacttgagg 120
atccaggaat ggcgtatttt ccttatatag catctgtctt aacagttttg tttgcgctac 180
_7_
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
ataaagcatc cattccaaca atgaaaattg cattgaaaac gtcaaaatgt tcatataaag 240
tggtgaaata ttgtattgta acaattttta atacgttgtt aaaattggca ggttataaag 300
agcagataactactaaagatgagatagaaaagcaaatggacagagtagtcaaagaaatga360
gacgccagctagaaatgattgacaaattgactacacgtgaaattgaacaagtagagttgc~420
ttaaacgcatttacgataaattgacggtgcaaacgacaggcgaaatagatatgacaaaag480
agatcaatcaaaaaaacgtgagaacgctagaagaatgggaaagtggaaaaaatccttatg540
aaccaagagaagtgactgcagcaatgtaagaggttgagctgccgtcgactgtcctcggaa600
gcggcggagttctttacagtaagcaccatcggacctgatggctgactgagaagccacagt660
cagccatatcgcgtgtggctcaagccttaatcccgtttaaccaatccggtcagcaccgga720
cgttaatggaaggaacggtcttaatgtgacc 751
<210> 17
<211> 751
<212> DNA
<213> Rotavirus Strain ALA
<400>
17
ggcttttaaaagttctgttccgagagagcgcgtgcggaaagatggataaacttaccgacc60
tcaattacacattgagcgtaatcactttaatgaatagtacattgcatgcaatattggaag120
atccagggatggcgtatttcccatacatagcatctgtgttgactgttctgttcactttac180
ataaagcatcaattccaacaatgaaaattgcgttaaaaacatctagatgttcctacaaag240
ttattaaatattgcattgtaaccatatttaatacattgttgaaattagctggatataaag300
aacaaataactactaaagatgaaattgaaaaacagatggatagagtaatcagagaaatga360
gacgtcagttggaaatgattgataaattgacaactcgtgaaattgaacaggtagaactac420
taagacgtatatatgacagattaacggtacgaaagactgatgagatagatatgtcgaagg480
agatcaatcagaaaaatatacgaacgctagatgaatgggagaatggaaaaaatccatatg540
aaccaagcgaagtgaccgcatcattgtgagaggttggactgccgtcgactgtctctggaa600
gcggcggagtccttcacagtaagtcccatcggacctgatgactggctgagaagccacagt660
cagtcatatcgcgtgtggctcaagccttaatcccgtttaaccaatccggtgagcgccgga720
cgttaatgga aggaatggtc ttagtgtgac c 751
<210> 18
<211> 751
<212> DNA
<213> Zapine Rotavirus Strain C-11
<400> 18
_g_
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
ggcttttaaaagttctgttccgagagagcgcgtgcggaaagatggataaacttaccgacc60
tcaattacacattgagcgtgatcactttaatgaatagtacattgcatacaatattggaag120
atccagggatggcgtatttcccatacatagcatctgtgttgactgttctgttcactttgc180
ataaagcatcaattccaacaatgaaaattgcgttaaaaacatctagctgttcctacaaag240
ttattaaatattgtcttgttactatatttaatacattgcctaaattagctggatataaag300
aacaaataactactaaacgtgaaattgaaaaacagatggatagagttatcagagaaatga360
gacgtcagttagaaatgattgataaattgacaactcgtgaaattgaacaggtagaactac420
taagacgtatatatgacaaattaacggtacgaaagactgatgagataggtatgttgaagg480
agatcaatcagaaaaatatacggacgctagatgaatgggagaatggaaagaatccatacg540
aaccaagcaaagtgaccgcatcattgtgagaggttggactgccgtcgactgtcctggaag600
cggcggagtccttcacagtaagtcccatcggacctgatgactggctgagaagccacagtc660
atatcatatcgcgtgtggctcaagccttaatcccgtttaaccaatccggtgagcgccgga720
cgttaatgga aggaatggtc ttagtgtgac c 751
<210> 19
<211> 751
<212> DNA
<213> Lapine Rotavirus Strain R-2
<400>
19
ggcttttaaaagttctgttccgagagagcgcgtgcggaaagatggaaaagcttaccgacc60
tcaactatacattgaatgtgatcactttattgaacagtacattgcatacaatattggagg120
atccagggatggcgtactttccttacattgcatctgtcctaacagttttattcacattac180
acaaagcgtcgattccaacgatgaaaattgccttaagaacatcaaaatgttcctataaag240
tgataaagtattgtattgtaacaattttcaatacgctactaaagttagccggctataaag300
aacagattactactaaagaatggattgaaaaacagttggacaaagtaataaaagaaatga360
gacgtcagctagaaatgatagataaattgacaactcgagaaattgaacaggtagagctac420
taaaacgtatatacgacaaactaatgatacgaaagactgatgaaatagatatgacgaagg480
agatcaatcaaaaaaatgtaaaaacgctagatgaatgggagaatgggaagaatccatatg540
aatcaaaagaagtgactgcagcaatgtaagaggttgggctgccgtcgactgtcttcggaa600
gcggcggagttcttcacagtaagttccatcggacctgatgagtggctgagaagccacagt660
cagtcatatcgcgtgtggctcaagccttaatcccgtttaaccaatccggtgagcgccgga720
cgttaatggaaggaagggtcttagtgtgacc 751
<210> 20
-9-
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
<211> 751
<2l2> DNA
<213> Lapine Rotavirus Strain BAP-2
<400> 20
ggcttttaaa agttctgttc cgagagagcg cgtgcggaaa gatggataaa cttaccgacc 60
tcaattacac attgagcgta atcactttaa tgaatagtac attgcatgca atattggaag 120
atccagggat ggcgtatttc ccatacatag catctgtgtt gactgtactg ttcactttac 180
ataaagcatc aattccaaca atgaaaattg cgttaaaaac atctagatgt tcctacaaag 240
ttattaaata ttgcattgta accatattta atacattgtt gaaattagct ggatataaag 300
aacaaataactactaaagatgaaattgaaagacagatggacagagtagtccgagaaatga360
gacgtcagttggaaatgattgataaattgacaacacgtgaaattgaacaggtagaactac420
taagacgtatatacgacagactaacggtgcgaaagactgatgagatagatatgtcgaagg480
agatcaatcagaaaaatatacggacgttagatgaatgggagaatggaaaaaatccatatg540
aaccaagcgaggtgaccgcatcattgtgagaggttggactgccgtcgactgtccctggaa600
gcggcggagtcctttacagtaagtcccatcggacctgatgactggctgagaagccacagt660
cagtcatatcgcgtgtggctcaagccttaatcccgcttaaccaatccggtgagcgccgga720
cgttaatggaaggaatggtcttagtgtgacc 751
<210> 21
<211> 751
<212> DNA
<2l3> Lapine Rotavirus Strain BAP (wildtype)
<400>
21
ggcttttaaaagttctgttccgagagagcgcgtgcggaaagatggataaacttaccgacc60
tcaattacacattgagcgtaatcactttaatgaatagtacattgcatgcaatattggaag120
atccagggatggcgtatttcccatacatagcatctgtgttgactgtactgttcactttac180
ataaagcatcaattccaacaatgaaaattgcgttaaaaacatctagatgttcctacaaag240
ttattaaatattgcattgtaaccatatttaatacattgttgaaattagctggatataaag300
aacaaataactactaaagatgaaattgaaaagcagatggacagagtaatccgagaaatga360
gacgtcagttggaaatgattgataaattgacaactcgtgaaattgaacaggtagaactac420
taagaagaatatacgacagactaacggtacgtaagactgatgagatagatatgtcgaagg480
aaatcaatcagaaaaatatacggacgttagatgaatgggagaatggaaaaaatccatatg540
aaccaagcgaggtgaccgcatcattgtgagaggttggactgccgtcgactgtccctggaa600
gcggcggagtccttcacagtaagtcccatcggacctgatgactggctgagaagccacagt660
cagtcatatcgcgtgtggctcaagccttaatcccgtttaaccaatccggtgagcgccgga720
-10-
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
cgttaatgga aggaatggtc ttagtgtgac c 751
<210> 22
<211> 750
<212> DNA
<213> Procine Rotavirus Strain A253
<400> 22
ggcttttaaa agttctattt cgagagagcg cgtgcggaaa gatggataag cttgcagacc 60
ttaattatac tttgagcgtt atcactttaa tgaatgatac actacactct ataattcaag 120
atccagggat ggcgtacttc ccatatattg catctgtact gactgtatta tttactctac 180
ataaggcatcaattcccacaatgaaaattgcgttaaaaacgtcaaagtgttcgtacaaag240
taattaagtattgcatggttacaatcattaatactcttctgaagttggctggttacaagg300
aacaggttactactaaggacgaaattgaacaacagatggatagaattgtaaaagagatga360
gacgtcaactggaaatgattgataaattgactactcgtgaaattgaacaggtagaattac420
ttaaacgtatacacgataaattggtagttagacctgtagacgttatagacatgtcgaaag480
aatttaaccagaaaaatattagaacgctagacgaatgggaaagtgggaaaaatccatacg540
aaccctcggaagttactgcgtctatgtgagaggttgagttgccgtcgtctgtcttcggaa600
gcggcggaactcttcaccgcaagccccattggacacgatggtttactgacaaaccccagt660
caatcatttcgcgtgtagcacatccctaatcccgaataaccaatccagcgaatgttggac720
gttaatggaaggaatggtcttaatgtgacc 750
<210> 23
<211> 750
<212> DNA
<213> Porcine Rotavirus Strain A131
<400> 23
ggcttttaaa agttctgttt cgagagagcg cgtgcggaaa gatggataag cttgcagacc 60
ttaattacac tttgagcgtt attactttaa tgaatgacac actacattct attattcaag 120
atccagggat ggcgatcttc ccatatatag catctgtact gactgtatta tttactctac 180
ataaggcatcaatacccacaatgaaaattgcgttaaaaacgtcaaagtgttcgtataaag240
taataaagtactgcattgttacaattatcaatactcttctgaaattggctggttacaagg300
aacaggttactacaaaggatgaaattgaacaacagatggacagaatcattaaagagatga360
gacgtcaactggaaatgatagataagttgactactcgtgaaattgaacaggtagaattac420
ttaagcgtattcatgataagttggttgtaaggccagtagacgttattgacatgtcgaaa.g480
aatttaatcagaagaatatacgaacgcttgacgaatgggaaagtggaaaaaatccatacg540
-11-
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
aaccgtcgga agtaactgca tctatgtgag aggttgagtt accctcgtct gtatttggga 600
gcggcgggac tcttcatcgc aaaccacatt ggacacgatg gtttactgac aaaccccagt 660
caatcatatc gcgtgtagca cagccataat cccgtataac aaatcctgcg aatgttggac 720
gttaatggaa ggaatggtct taatgtgacc 750
<210> 24
<211> 750
<212> DNA
<2l3> Porcine Rotavirus Strain A411
<400>
24
ggcttttaaaagttctgttccgagagagcgcgtgcggaaagatggataagcttgacgatc60
ttaattatactttgagcgtcatcactttaatgaatgacacactacattctataattcaag120
atccaggaatggcgtacttcccatacatagcatctgtactgactgttttatttactctac180
ataaggcatcaattcccacaatgaaaattgcgttaagaacgtcaaagt'gttcgtataaag240
taataaaatactgcattgttacaatttttaatactcttctgaaattggctggttacaaag300
aacaggttactactaaagacgaaattgaacaacagatggacagaattatcaaagagatga360
gacgtcaactggaaatgattgacaaattgactactcgtgaaattgaacaggtagaattac420
ttaaacgtattcacgataaactggttgcaaggtcagttgacgttatagacatgtcgaaag480
aatttaatcagaaaaatataagaacgctagatgaatgggaaagtggaaaaaatccctacg540
aaccgtcggaagtaactgcatctatgtgagaggttgagttgccgtcatcagtctttggga600
gcggcggaactcttcatcgcaagccccattggacccgatggttgactgagaagccacagt660
caatcatttctcgtgtagcacagccctaatcccgattaaccaatccagcgaatgttggac720
gttaatggaaggaatggtcttaatgtgacc 750
<210> 25
<211> 675
<212> DNA
<213> Porcine Rotavirus Strain A34
<400> 25
gatggataag cttgccgacc tcaactacac attgagtgta atcactttaa tgaatgatac 60
gttacactct attattcaag atccaggaat ggcgtatttt ccatatatcg catctgttct 120
aactgtttta tttactctac ataaagcatc aattccaacg atgaaaatag cattaagaac 180
gtcaaaatgt tcatacaaag taattaaata ttgtatggtt acgatcatta atactcttct 240
aaagttggct ggttataaag aacaggttac taccaaggat gaaatcgaac aacagatgga 300
cagaattgtt aaagagatga gacgtcaact ggagatgatt gacaaattga caactcgtga 360
aattgaacag gtcgaattac ttaagcgtat acatgataaa ttagttacta gaccagttga 420
-12-
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
tgctatagacatgtcgaaagaatttaatca gaagaatatcagaacgctagatgaatggga480
aagcggaaaaaatccatatgaaccatcaga agtgactgcatctatgtgagaggttgagtt540
gccgtcgtctgtcttcggaagcggcggaac tcttcaccgcaagccccattggacctgatg600
gttgactgagaagccacagtcaatcatatc gcgtgtggctcagccttaatcccgtttaac660
caatccagcgaatgt 675
<210>
26
<211>
751
<212>
DNA
<213>
Equine
Rotavirus
Strain
H-2
<400>
26
ggcttttaaaagttctgttccgagagagcgcgtgcggaaagatggataagcttaccgacc60
tcaactatacattgaatgtgatcactttattgaacagtacattgcatacaatattggagg120
atccagggatggcgtactttccttacattgcatctgtcctaacagttttattcacattac180
acaaagcgtcgattccaacgatgaaaattgccttaagaacatcaaaatgttcgtataaag240
tgataaagtattgtattgtaacaattttcaatacgctactaaagttagcaggctataaag300
aacagattactactaaagatgaaatagaaaaacaaatggatagagtagttaaagaaatga360
gacgtcatttagagatgattgataaattgactacacgtgaaattgaacaagtagaattac420
ttaaacgtatttatgataaactgatgatacgggcaacagacgaaatagatatgacgaaag480
aaatcaatcaaaagaacgtgaaaacgctagaagaatgggaaaatggaaagaatccttatg540
aatcaaaagaagtgactgcagcaatgtaagaggttgagctgccgtcgactatcttcggaa600
gcggcggagttctttacagtaagctccatcagacctgatggctggctgagaagccacagt660
cagccatatcgcgtgtggctcaagccttaatcccgtttaaccaatccggtcagtaccgga720
cgttaatggaaggagtggtcttagtgtgaag 751
<210> 27
<211> 751
<212> DNA
<213> Equine Rotavirus Strain FI-23
<400> 27
ggcttttaaa agttctgttc cgagagagcg cgtgcggaaa gatggataag cttaccgacc 60
ttaattatac attgaatgta attactctat tgaacagtac attgcataca attttagagg 120
atccagggat ggcgtatttc ccttacattg catctgtact aacagtatta ttcacattac 180
acaaagcgtc gattccaacg atgaagattg ccttaagaac atcaaaatgt tcgtacaagg 240
tgattaagta ttgtatagtt acaattttca atacgctact aaagttagca ggctataagg 300
-13-
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
aacagattactactaaggacgaaatagaaaaacaaatggatagagttgttaaagaaatga360
ggcgtcacctagagatgatagataagttgactacacgtgaaatagagcaagttgaattac420
ttaaacgtatatacgataagctgatggcacgagcaacagatgaaattgatatgactaaag480
aaataaatcagaagaacgtgaaaaegttagaagaatgggaaaatggaaagaatccttacg540
aatcaaaacgaatgactgcagcaatgtaagaggttgaactgccgtcgactatctttggaa600
gcgggggggtactatatagtaagctccatcagacctaatagctggctgagaagccacagt660
cagcaatttaaaaagtggctcaagccttaattcccttcaaccaatccggtcagtaccgga720
cgttaatggaaggag~ggtcttagtgtgaag 751
<210>
28
<211>
751
<212>
DNA
<213>
Equine
Rotavirus
Strain
FI-14
<400>
28
ggcttttaaaagttctgttccgagagagcgcgtgcggaaagatggataaactaaccgacc60
tcaactatacattgaacgtaatcactttaattaacagcacattgcatacaattttagagg120
atcccggaatggcgtatttcccttacattgcatctgtattaacagtattattcacattac180
acaaggcatcgataccaacgatgaagatagccttgaaaacatcaaagtgttegtataaag240
tagtaaaatactgtatagttacaatttttaatacgctactaaaattagcaggctacaaag300
aacaaataactactaaagatgaaattgagaagcaaatggacagagtaattaaagaaatga360
gacgtcatttagagatgatagacaagttgacaactcgtgagatagagcaagttgaactac420
ttaagcgtatatacgataagctaatgattcgggctacggacgaaattgatatgtcgaaag480
aaattaaccaaaagaacgtaagaacgttagaagaatgggaaaacggaaagaatccttatg540
aatcaaaagaagttactgcagcaatgtaagaggttgagctgccgtcgactatcttcggaa600
gcggcggagtattttacagtaagctccaccaaacctgatggctggcagaaaaaccccatt660
cagcaatttcgcgtgtggctcataacttaattccgttcaatcactccggtcagtaccgga720
cgttaatggaaggagtggtcttagtgtgaag 751
<210> 29
<211> 751
<212> DNA
<213> Bovine Rotavirus Strain BRV033
<400> 29
ggctttaaaa agttctgttc cgagagagtg tgtgcgggaa gatggagaag cttaccgacc 60
tcaactacac atcgagtgtt atcactctaa tgaacaacac attgcatacg attcttgagg 120
accccggaat ggcgtacttc ccatacattg catctgtcct aacagttttg tttacgttgc 180
-14-
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
acaaggcatc tatacctaca atgaagatag cactgaaaac gtccaagtgt tcatacaaag 240
tagtaaaatactgtatagtaacgatattcaatacgttgttgaaattggcaggttacaaag300
aacagataactactaaagatgagatagaaaagcaaatggacagggttgttaaagagatga360
gacgtcagtttgaaatgattgataagttgactacacgtgaaatagagcaggtagagttgc420
taaagcgcatacacgacaagttgatggttcgagcaacagatgagattgatatgacgaagg480
aaataaaccaaaagaacgtaagaacgctagaagaatgggaaaatggaaaaaatccttatg540
aacccaaggaggtgactgcagcgatgtaagaggttgagctgccctcgactgtcttcggaa600
gcggcggagttcttcacagtaagccacatcggacatgatgacttactgaaaagccccagt660
cagtcatttcccgagtggcttaagccttaatccccttcaaccattcaggtcagcaccgga720
cgttaatggagggaacggtcttaatgtgaca 752
<210> 30
<211> 751
<212> DNA
<213> Bovine Rotavirus Strain B223
<400>
30
ggctttaaaaagttctgttccgagagagtgtgtgcgggaagatggaaaagctaaccgacc60
tcaactatacattgagtgttatcactctaatgaactccacattgcatacgattcttgagg120
accccgggatggcgtacttcccatatattgcatcagttttaacagtattattcacgttgc180
acaaggcatctatacccacaatgaagattgctctaaagacgtccaagtgttcatacaaag240
tagtaaaatattgcattgttacgattttcaatacgttgttgaaattggctgggtacaaag300
aacagataactactaaagatgagatagagaaacagatggaaagggtagtaaaggaaatga360
gacgtcacttcaaaatgatagacaaattgacaactcgtgaaattgagcaggtaggattgc420
taaagcgcattcacgacaagttggatatacgggctgttgatgaaatagacatgacgaaag480
aaattaaccagaaaaacgttagaacgctagaagaatgggagtggggaaaaaatccctatg540
aacccaaagaagttactgctgcaatgtaagaggttgagctaccttcgacagtattcggaa600
gcgggggggtactacacagtaagcctcaacggttatgttgactaactgagaaacctcaat660
cagtcatttccagagttttttaagccttaatccccttcaaccattcaggtcagcaccgga720
cgttaatggaaggaacggtcttaatgtgaca 751
<210> 31
<211> 750
<212> DNA
<213> Canine Rotavirus Strain CU-1
<400> 31
-15-
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
ggcttttaaa agttctgttc cgagaaagcg catgcggaaa gatggagaag cttgcagacc 60
tcaactatac cctgagtgta atcacgctaa tgaatgatac tttgcacact attatggagg 120
atcccggaat ggcatacttc ccatatattg catctgttct aactgtacta tttacattac 180
ataaggcatcaatcccaaccatgaaaatcgcacttaaaacatcaagatgttcatacaagg240
ttatcaagtactgcatagtatcagtatttaacactctattgaagttggctggatacaaag300
agcagataactactaaagatgaaatagaaaaacaaatggacagagttgttaaagaaatga360
ggcgtcagctggaaatgattgataaactaaccacaagggagatagaacaggttgaacttc420
ttaaacgaatacacgatatgttaattgcaaagcccgtagacaagatagatatgtcgcaag480
agttcaaccaaaagcatttcaaaacactaaacgagtgggcagagggtgaaaatccatacg540
aaccgagagaagtaactgcatctttatgagaggttgaactgccgtcttcggtatgcggga600
gcggaggagtaataaacagaaaatctcatcgaacttgatgaatggtagagaaacctcatt660
cagtaatttcgcgggtgacttagtcttattcacgttttaccattccagccagtgctggac720
gttaatggaaggaatggtcttaatgtgacc 750
<210> 32
<211> 750
<212> DNA
<213> Porcine Rotavirus A
<400> 32
ggcttttaaa agttctgttc cgagagagcg cgtgcggaaa gatggataag cttgccgacc 60
tcaattacac attgagcgta atcactttaa tgaatgacac actacactct attattcaag 120
atccaggaatggcgtattttccatatattgcatctgttctgactgttttatttactctac180
ataaagcatcaattccaacaatgaaaatagcgttaaaaacgtcaaagtgttcgtacaaag240
taattaaatattgcatggttacaatcattaatactcttctgaagttggctggttataaag300
aacaggttactactaaggatgaaattgaacaacagatggacagaattattaaagagatga360
gacgtcaactggaaatgattgacaaattgacgactcgtgaaattgaacaggttgaattac420
ttaaacgtatacatgacaaattagctgctagatcagttgacgctatagatatgtcgaaag480
aatttaatcagaaaaatattcgaacgctagatgaatgggaaagtggaaaaaatccatatg540
aaccgtcggaagtaactgcgtctatgtgagaggttgagttgccgtcgtctgtcttcggaa600
gcggcggaactcttcaccgcaagccccattggacccgatggttgactgagaagccacagt660
caatcatatcgcgtgtggctcagccttaatcccgtttaaccaatccagcgaatgttggac720
gttaatggaaggaatggtcttaatgtgacc 750
<210> 33
-16-
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
<211>
528
<212>
DNA
<213>
Simian
11 Rotavirus
(strain
SA 11)
<400>
33
atggaaaagcttaccgacctcaattatacattgagtgtaatcactctaatgaacaataca60
ttgcacacaatacttgaggatccaggaatggcgtattttccttatatagcatctgtctta120
acagttttgtttgcgctacataaagcatccattccaacaatgaaaattgcattgaaaacg180
tcaaaatgttcatataaagtggtgaaatattgtattgtaacaatttttaatacgttgtta240
aaattggcaggttataaagagcagataactactaaagatgagatagaaaagcaaatggac300
agagtagtcaaagaaatgagacgccagctagaaatgattgacaaattgactacacgtgaa360
attgaacaagtagagttgcttaaacgcatttacgataaattgacggtgcaaacgacaggc420
gaaatagatatgacaaaagagatcaatcaaaaaaacgtgagaacgctagaagaatgggaa480
agtggaaaaaatccttatgaaccaagagaagtgactgcagcaatgtaa 528
<210> 34
<211> 1356
<212> DNA
<213> Rotavirus subgroup 1 (VP6)
<220>
<221> misc_feature
<222> (1340)..(1356)
<223> unknown
<400>
34
ggcttttaaacgaagtcttcaacatggatgtcctgtactcattgtcaaaaactcttaaag60
acgccagagacaagatcgttgaaggcacattatactccaatgtaagtgatctaattcaac120
agtttaatcaaatgataattactatgaatggaaatgaatttcaaactggaggaattggca180
acttacccattagaaattggaattttgattttggcctacttggaactactctactaaact240
tagatgctaattacgttgaaactgcacgtaatacaattgattattttgtcgattttgtgg300
ataatgtatgcatggatgagatggttagggaatcacaaagaaatggaatcgcaccgcaat360
cagactcactcagaaaactgtcaggcattaaatttaaaagaataaattttgacaattcat420
cagagtatattgaaaattggaatctgcagaatagaagacagagaacaggtttcacattcc480
acaagccgaacatctttccttattcagcatcatttacactaaaccgatcgcaaccagctc540
atgataatttaatgggtacaatgtggttaaatgcaggatcagaaattcaggttgctggat600
ttgattattcatgtgctattaacgctccagctaatacacaacaatttgaacatattgtac660
agctccggagagtactaactactgctacgataactcttttaccagacgcagaaagattta720
gttttccaagagtgattaattcagctgacggagcaactacatggtattttaatccagtga780
-17-
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
ttcttaggcc aaataacgtt gaagtagagt ttctgttgaa tgggcaaata ataaacactt 840
atcaagcaag atttggaaca ataatagcta gaaattttga tactattaga ttgtcattcc 900
agctaatgag accaccaaat atgacaccaa cagtagctgc actatttcca aatgcacaac 960
catttgaaca tcatgctacg gtaggcctaa cactaagaat tgagtctgca gtgtgtgaat 1020
cagtactagctgatgcgagcgaaacaatgctagctaatgtaacgtctgttaggcaggaat1080
acgcaataccagttggaccagtatttccaccaggcatgaattggactgatctaatcacta1140
attactcaccatctagggaggataatttgcagcgtgtgtttacagtggcttccattagaa1200
gcatgctaattaaatgaggaccaagctaactacttggtatccgaactttataagcatgta1260
gctatgtcaagctatttgaactttgtaagtaaggatgtatttatacattcgctacacaaa1320
gtaatcactt caatgatgtn nnnnnnnnnn nnnnnn 1356
<210> 35
<211> 1356
<212> DNA
<213> Rotavirus subgroup 2
<400>
35
ggctttaaaacgaagtcttcgacatggaggttctgtactcactgtcaaaaactcttaaag60
atgctagggacaaaattgttgaaggtacattatattctaatgttagcgatcttattcagc120
aattcaatcaaatgatagtaactatgaatggaaatgattttcagactggaggaattggta180
atttacctgttagaaattggactttcgattttggtctattaggtacaacacttttgaact240
tggatgctaattatgttgagaatgcaagaactataattgaatattttattgactttattg300
ataatgtatgtatggatgaaatggcaagagaatctcaaagaaatggagtagcgccacaat360
ctgaagcgttgagaaagttagcgggaattaaatttaagagaataaatttcgataattcat420
cagaatacatagaaaattggaacttacaaaatagaagacagcgcaccggatttgtttttc480
ataaacctaacatatttccatactcagcttcatttactctaaatagatctcaaccaatgc540
atgataatttaatgggaaccatgtggcttaatgctggatcagaaattcaagtggctggat600
ttgactactcatgcgccataaatgcaccagcgaacatacagcaatttgaacatatcgtcc660
agcttaggcgcgcactgactacagctactataactttattacctgatgcagagagattta720
gttttccaagagtaattaattcagctgatggcgcgactacatggttctttaatccagtta780
ttctaagaccaaacaatgtagaggtagaatttttgttgaatggacaaattattaatacat840
atcaggctagatttggtactatcatcgcaagaaattttgatgcaattcgtttattatttc900
agttgatgcg tccacctaat atgacaccag ctgttaatgc actgtttcca caagcacaac 960
cttttcagca ccatgcaaca gttggactta cattacgtat tgaatctgcg gtttgtgaat 1020
-18-
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
cagtgcttgcggacgcaaatgaaactctgttagcaaatgtgaccgcggtg cgtcaagaat1080
atgccataccagttggaccggtatttccaccaggcatgaattggactgaa ttaattacta1140
actattcgccatctagagaagataacttgcaacgcgttttcacggtagct tccattagaa1200
gcatgttgattaagtgaggaccagactaagcatctggtatccaatcttag ttagcatgta1260
gctacatcaagtcattcagactcttcaagtaaggacatgatttcatgttc gctacgtaga1320
gtaactgtctgaatgatgtagtgagaggatgtgacc 1356
<210>
36
<211>
2717
<212>
DNA
<213>
Bovine
rotavirus
(VP2)
<400>
36
ggctattaaaggctcaatggcgtacaggaagcgcggagctaaacgtgaaaacttaccaca60
acaaaatgaacgtctgcaagaaaaagaaattgaaaaagatgtggatgtaactatggagaa120
taaaaataacaatagaaagcagcaattatctgataaagtactatcacaaaaagaggaaat180
aataactgatgcacaagatgatattaaaatagctggtgagattaaaaaatcatcaaaaga240
agagtcaaaacagttgctcgaaatattaaaaacgaaagaagaccatcagaaagaaataca300
gtatgaaattctacaaaaaacgataccgacttttgaatcaaaagaatcaattttgaaaaa360
attagaagatat.aagaccggagcaagctaagaagcaaatgaaattgtttagaatatttga420
accaaaacaattaccaatctatcgagcaaatggtgagaaagaattgagaaatagatggta480
ttggaaattgaaaaaggatacgctgccagatggagattatgatgtacgagaatatttctt540
aaatttatatgatcagatcctgatagaaatgccagattatttgctactgaaagatatggc600
tgtagaaaataaaaactctagagatgctggtaaagttgtagattctgaaacggcaaatat660
ttgtgatgctatatttcaagatgaagagacagagggagttgtcagaagattcattgcaga720
tatgagacaacaggttcaggctgatagaaatattgtcaattatccatcaattttacatcc780
aattgatcacgcatttaatgaatattttctaaatcatcaattagtcgaaccactaaataa840
tgaaatcatttttaattatataccagaaagaataaggaatgatgttaactatattttgaa900
tatggatatgaatttgccatcaacagcaagatatattagaccaaatttattgcaagatag960
actaaatttacatgataattttgaatcattatgggacacaataactacatcaaattatat1020
actagccagatcagttgtgcctgatttgaaggaaaaagaattagtttcaactgaagctca1080
gatacagaaaatgtctcaagatttgcaacttgaagctttaacgatacaatctgaaacgca1140
gtttcttgctggcataaattcacaagcagcaaatgattgttttaaaacattgatagcagc1200
tatgttaagccagcgtacaatgtcattagattttgtaaccacgaattatatgtcacttat1260
-19-
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
atctggtatgtggctattgaccgttataccaaatgatatgtttcttcgtgaatcattagt1320
cgcatgcgaattggccataataaatactatagtttatccagcatttggaatgcaaagaat1380
gcattatagaaatggtgatccccagactccgtttcaaatagcagaacagcaaatacaaaa1440
ttttcaagtagctaattggttacattttattaataataatagatttaggcaagttgttat1500
tgatggagtgttaaatcaaacacttaacgataatattaggaatggacaagttattaatca1560
gttaatggaagcattaatgcagctatctagacaacaatttccgactatgccagttgatta1620
taaaagatcaatccaaagaggaatattactattatctaacagattaggtcagttagttga1680
tttaacaagattagtatcatataattatgaaactctaatggcttgtgtaactatgaatat1740
gcaacatgttcaaactctcactaccgaaaaattacaattaacttctgtcacatctttatg1800
tatgttaattggaaatactacagtaattccaagtccacaaacattatttcactattataa1860
cataaatgta aattttcatt caaattataa cgaacgaatt aacgacgcag tggctatcat 1920
tacggctgct aatagactaa acttatatca gaaaaaaatg aaatcaatag ttgaggattt 1980
tttgaaaaga ttgcaaattt ttgatgtacc acgagtacca gatgaccaaa tgtacaggtt 2040
gagagataga cttaggttat taccagttga aagacgaaga cttgatatat ttaatttaat 2100
attaatgaatatggagcagatcgaacgagcttcagataaaattgctcaaggagtaataat2160
tgcttatagagatatgcaactagaaagagatgagatgtatggatatgtcaacattgctag2220
aaatctcgatggatatcaacaaattaacctagaggagttgatgagaactggagactatgg2280
gcaaattactaatatgttattaaacaatcagcctgtagctttagtaggggcattaccatt2340
tgtgacggattcttcagttatatcactcattgcaaaattagatgctacagtttttgctca2400
aatagttaaacttagaaaagtggacactttaaaaccaatattgtataagataaattctga2460
ttctaatgatttctacttagttgcaaattatgattggataccaacttcaaccacaaaagt2520
ctataaacaagtaccacaaccttttgatttcagagcgtcaatgcatatgttaacgtctaa2580
tttgacttttacagtttattctgatttattatctttcgtttctgcagacacggttgaacc2640
cattaacgcagttgcttttgacaatatgcgcattatgaacgaactgtaaacgccaacccc2700
actgtggagatatgacc 2717
<210> 37
<21l> 2690
<212> DNA
<213> Simian rotavirus SA11 clone 3 (VP2)
<400> 37
ggctattaaa ggctcaatgg cgtatcgaaa acgtggagcg cgtcgtgaga cgaatctaaa 60
acaagatgaa cgaatgcaag aaaaagaaga tagcaagaac attaataatg acagtcctaa 120
-20-
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
atcacaatta tcagaaagag tattatctaa gaaagaagag ataattacag ataatcaaga 180
agaagttaag atatctgatg aggtaaaaaa atctaataaa gaagaatcga aacagttgtt 240
agaagtactt aaaacaaaag aggaacatca aaaagaagtt cagtatgaaa tattacaaaa 300
aactatccct acatttgaac caaaagagtc aatactcaaa aaattagaag acataaaacc 360
agaacaagcaaagaaacaaactaaactgtttcgaatatttgaaccgaaacaattgcctat420
ttatagagctaatggagaaagagagcttcgtaatagatggtattggaaattgaaacgaga480
tactcttcctgatggagattatgatgttagagagtattttttaaatttatatgatcaagt540
attaatggaaatgccggattatctattacttaaagatatggctgtagagaataaaaattc600
aagggatgctggcaaagtagttgattctgaaacagccgcaatatgcgatgctatttttca660
agatgaagaaccgaaggcagtaagaagattcatagctgagatgagacaacgagttcaagc720
tgatcgaaatgtagtcaattatccatctatattgcatccaattgaccatgcgtttaacga780
atacttcttacaacatcagttggtagaaccattaaataatgtatacattttcaattacat840
accagagagaataagaaatgatgtcaactatatattaaatatggacaggaatttaccgtc900
tactgctagatatatcagaccaaacttgctacaagataggttaaatttacatgataattt960
tgagtcactctgggatactataactacatctaattatattttagcaagatctgt,ggtgcc1020
agacctaaaagaattagtatctactgaggcacaaatccagaaaatgtcacaagatttgca1080
attggaagctttgacaatacaatcagagactcagtttttaacaggtataaactcacaagc1140
agctaatgattgttttaaaactttgattgctgctatgttgagtcagagaaccatgtcatt1200
agatttcgtaacgacaaattacatgtcacttatttcaggcatgtggttactcactgtgat1260
tccaaatgatatgtttataagagaatcattagtagcatgtcaactagccataataaatac1320
cattgtttatccggcattcggaatgcaaagaatgcattataggaatggtgatccacagac1380
tccctttcaaattgcagagcaacagattcaaaattttcaggtagctaattggttacattt1440
tgttaattataatcagtttagacaagtagtgattgatggagtgttaaatcaagtcttgaa1500
tgataatataagaaatggtcatgtagtcaaccaattaatggaagctctgatgcaattatc1560
tagacaacagtttcccacaatgccagttgattataaaagatctatacagagaggaatttt1620
gctgctttctaacagacttggtcagcttgtcgatttaacaagattgttatcatacaatta1680
tgagacattaatggcatgcataacaatgaatatgcagcatgttcaaacattaacaactga1740
aaaattgcaattaacatcagtaacatcattatgtatgctaattggaaatgctacggttat1800
accgagtccgcaaacattgtcccattactataatgtgaatgtcaattttcattcaaatta1860
taatgaaagaattaatgacgcagttgcaattataactgcggcaaatagattaaatttata1920
-21-
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
tcaaaagaaaatgaaatcaatagttgaggactttctgaaaagattacagatatttgatgt1980
tgcgagagtaccagatgaccaaatgtatagattgagagatagattaagactattaccagt2040
tgaaataagaagattagatatttttaatttgatagcaatgaatatggaacagattgaacg2100
tgcatcagataaaattgcacaaggagttataatagcataccgagatatgcagttagaacg2160
agatgagatgtatggttacgtcaatattgccagaaacttggacggatttcaacaaataaa2220
tcttgaagaattgatgagatcaggagattatgctcaaattactaacatgctacttaataa2280
tcaaccagtagctttagttggagcgctaccatttataacggattcatcagtgatttcgtt2340
aatagctaaactagatgcaaccgtttttgcacagattgtcaaacttagaaaggtcgacac2400
gttaaaacccatcctatataagataaattcagattctaatgacttttatttggtggctaa2460
ttatgattggattcctacatctactacaaaagtgtataaacaagttccacaacaatttga2520
ttttagagcgtcaatgcatatgttaacgtctaacctaacatttaccgtatattcagattt2580
gcttgcgttcgtttcagctgatactgttgaaccaattaatgctgttgcttttgataatat2640
gcgcatcatgaacgaactgtaaacgccaaccccattgtggagatatgacc 2690
<210> 38
<211> 1356
<212> DNA
<213> Simian rotavirus SA 11 clone 3 (VP6)
<400>
38
ggcttttaaacgaagtcttcaacatggatgtcctatactctttgtcaaagactcttaaag60
acgctagagacaaaattgtcgaaggcacattgtattctaacgtgagtgatctaattcaac120
aatttaatcaaatgataattactatgaatggaaatgaatttcaaactggaggaatcggta180
atttgccaattagaaactggaattttaatttcgggttacttggaacaactttgctgaact240
tagacgctaattatgttgaaacggcaagaaatacaattgattatttcgtggattttgtag300
acaatgtatgcatggatgagatggttagagaatcacaaaggaacggaattgcacctcaat360
cagactcgctaagaaagctgtcagccattaaattcaaaagaataaattttgataattcgt420
cggaatacatagaaaactggaatttgcaaaatagaagacagaggacaggtttcacttttc480
ataaaccaaacatttttccttattcagcatcatttacactaaatagatcacaacccgctc540
atgataatttgatgggcacaatgtggttaaacgcaggatcggaaattcaagtcgctggat600
ttgactactcatgtgctattaacgcaccagccaatatacaacaatttgagcatattgtgc660
cactccgaagagtgttaactacagctacgataactcttctaccagacgcggaaaggttta720
gttttccaagagtgatcaattcagctgacggggcaactacatggtttttcaacccagtga780
ttctcaggccgaataacgttgaagtggagtttctattgaatggacagataataaacactt840
-22-
CA 02427809 2003-05-02
WO 02/36172 PCT/USO1/45255
atcaagcaag atttggaact atcgtagcta gaaattttga tactattaga ctatcattcc 900
agttaatgag accaccaaac atgacaccag cagtagcagt actattcccg aatgcacagc 960
cattcgaaca tcatgcaaca gtgggattga cacttagaat tgagtctgca gtttgtgagt 1020
ctgtactcgccgatgcaagtgaaactctattagcaaatgtaacatccgttaggcaagagt1080
acgcaataccagttggaccagtctttccaccaggtatgaactggactgatttaatcacca1140
attattcaccgtctagggaggacaatttgcaacgcgtatttacagtggcttccattagaa1200
gcatgctcattaaatgaggaccaagctaacaacttggtatccaactttggtgagtatgta1260
gctatatcaagctgtttgaactctgtaagtaaggatgcgtatacgcattcgctacactga1320
gtaatcactctgatggtatagtgagaggatgtgacc 1356
<210>
39
<211>
788
<212>
DNA
<213> 94-881
Rotavirus
vp2
<400>
39
caagaacattaataatgacagtcctaaatcacaattatcagaaagagtattatctaagaa 60
agaagagataattacagataatcaagaagaagttaagatatctgatgaggtaaaaaaatc 120
taataaagaagaatcgaaacagttgttagaagtacttaaaacaaaagaggaacatcaaaa 180
agaagttcagtatgaaatattacaaaaaactatccctacatttgaaccaaaagagtcaat 240
actcaaaaaattagaagacataaaaccagaacaagcaaagaaacaaactaaactgtttcg 300
aatatttgaaccgaaacaattgcctatttatagagctaatggagaaagagagcttcgtaa 360
tagatggtattggaaattgaaacgagatactcttcctgatggagattatgatgttagaga 420
gtattttttaaatttatatgatcaagtattaatggaaatgccggattatctattacttaa 480
agatatggctgtagagaataaaaattcaagggatgctggcaaagtagttgattctgaaac 540
agccgcaatatgcgatgctatttttcaagatgaagaaccgaaggcagtaagaagattcat 600
agctgagatgagacaacgagttcaagctgatcgaaatgtagtcaattatccatctatatt 660
gcatccaattgaccatgcgtttaacgaatacttcttacaacatcagttggtagaaccatt 720
aaataatgtatacattttcaattacataccagagagaataagaaatgatgtcaactatat 780
attaaata 788
-23-
